Monitoring hybridization during PCR using SYBR™ Green I

ABSTRACT

The present invention is directed to a method and kits for monitoring a nucleic acid during amplification. More particularly, the present invention relates to a method wherein the nucleic acid is monitored during polymerase chain reaction using a double-stranded nucleic acid binding dye capable of producing a fluorescent signal related to the amount of the nucleic acid present in a sample, wherien the dye is selected from the group consisting of SYBR(TM) Green I and pico green.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is continuation of U.S. application Ser. No.09/635,344, filed Aug. 9, 2000, U.S. Pat. No. 6,232,079, which is adivisional of U.S. application Ser. No. 08/869,276, filed Jun. 4, 1997,now U.S. Pat. No. 6,174,670, which is a continuation-in-part of U.S.application Ser. No. 08/818,267, filed Mar. 17, 1997, abandoned, whichis a continuation-in-part of U.S. application Ser. No. 08/658,993, filedJun. 4, 1996, now abandoned. Each of the above-identified applicationsare now each individually incorporated herein by reference in theirentireties.

MICROFICHE APPENDIX

A microfiche appendix is contained in U.S. application Ser. No.08/869,276, herein incorporated by reference.

BACKGROUND OF THE INVENTION

This invention relates generally to observing fluorescence signalsresulting from hybridization in conjunction with the polymerase chainreaction. More specifically, the present invention relates to observinghybridization with fluorescence during and/or immediately after PCR andusing this information for product identification, sequence alterationdetection, and quantification.

The polymerase chain reaction (PCR) is fundamental to molecular biologyand is the first practical molecular technique for the clinicallaboratory. Despite its usefulness and popularity, current understandingof PCR is not highly advanced. Adequate conditions for successfulamplifications must be found by trial and error and optimization isempirical. Even those skilled in the art are required to utilize apowerful technique without a comprehensive or predictive theory of theprocess.

PCR is achieved by temperature cycling of the sample, causing DNA todenature (separate), specific primers to attach (anneal), andreplication to occur (extend). One cycle of PCR is usually performed in2 to 8 min, requiring 1 to 4 hours for a 30-cycle amplification. Thesample temperature response in most PCR instrumentation is very slowcompared to the times required for denaturation, annealing, andextension. The physical (denaturation and annealing) and enzymatic(extension) reactions in PCR occur very quickly. Amplification times forPCR can be reduced from hours to less than 15 min. Incorporated hereinby reference in their entireties are each of the following individualapplications, which disclose such a rapid cycling system: U.S.application Ser. No. 08/818,267, filed Mar. 17, 1997, entitled Methodfor Detecting the Factor V Leiden Mutation, which is acontinuation-in-part of U.S. patent application Ser. No. 08/658,993,filed Jun. 4, 1996, entitled System And Method For Monitoring PCRProcesses, which is a continuation-in-part of U.S. patent applicationSer. No. 08/537,612, filed Oct. 2, 1995, entitled Method For RapidThermal Cycling of Biological Samples, which is a continuation-in-partof U.S. patent application Ser. No. 08/179,969, filed Jan. 10, 1994,(now U.S. Pat. No. 5,455,175), entitled Rapid Thermal Cycling Device,which is a continuation-in-part of U.S. patent application Ser. No.07/815,966 filed Jan. 2, 1992, (now abandoned) entitled Rapid ThermalCycling Device which is a continuation-in-part of U.S. patentapplication Ser. No. 07/534,029 filed Jun. 4, 1990, (now abandoned)entitled Automated Polymerase Chain Reaction Device. The copending U.S.application filed in the U.S. Patent and Trademark Office on Jun. 4,1997, entitled System and Method for Carrying Out and MonitoringBiological Processes as Ser. No. 08/869,275 and naming Carl T. Wittwer,Kirk M. Ririe, Randy P. Rasmussen, and David R. Hillyard as applicants,is also hereby incorporated by reference in its entirety. Rapid cyclingtechniques are made possible by the rapid temperature response andtemperature homogeneity possible for samples in high surfacearea-to-volume sample containers such as capillary tubes. For furtherinformation, see also: C. T. Wittwer, G. B. Reed, and K. M. Ririe, Rapidcycle DNA amplification, in K. B. Mullis, F. Ferre, and R. A. Gibbs, Thepolymerase chain reaction, Birkhauser, Boston, 174-181, (1994). Improvedtemperature homogeneity allows the time and temperature requirements ofPCR to be better defined and understood. Improved temperaturehomogeneity also increases the precision of any analytical techniqueused to monitor PCR during amplification.

Fluorimetry is a sensitive and versatile technique with manyapplications in molecular biology. Ethidium bromide has been used formany years to visualize the size distribution of nucleic acids separatedby gel electrophoresis. The gel is usually transilluminated withultraviolet light and the red fluorescence of double stranded nucleicacid observed. Specifically, ethidium bromide is commonly used toanalyze the products of PCR after amplification is completed.Furthermore, EPA 0 640 828 A1 to Higuchi & Watson, hereby incorporatedby reference, discloses using ethidium bromide during amplification tomonitor the amount of double stranded DNA by measuring the fluorescenceeach cycle. The fluorescence intensity was noted to rise and fallinversely with temperature, was greatest at the annealing/extensiontemperature (50° C.), and least at the denaturation temperature (94°C.). Maximal fluorescence was acquired each cycle as a measure of DNAamount. The Higuchi & Watson application does not teach usingfluorescence to monitor hybridization events, nor does it suggestacquiring fluorescence over different temperatures to follow the extentof hybridization. Moreover, Higuchi & Watson fails to teach or suggestusing the temperature dependence of PCR product hybridization foridentification or quantification of PCR products.

The Higuchi & Watson application, however, does mention using otherfluorophores, including dual-labeled probe systems that generateflourescence when hydrolyzed by the 5′-exonuclease activity of certainDNA polymerases, as disclosed in U.S. Pat. No. 5,210,015 to Gelfand etal. The fluorescence observed from these probes primarily depends onhydrolysis of the probe between its two fluorophores. The amount of PCRproduct is estimated by acquiring fluorescence once each cycle. Althoughhybridization of these probes appears necessary for hydrolysis to occur,the fluorescence signal primarily results from hydrolysis of the probes,not hybridization, wherein an oligonucleotide probe with fluorescentdyes at opposite ends thereof provides a quenched probe system usefulfor detecting PCR product and nucleic acid hybridization, K. J. Livak etal., 4 PCR Meth. Appl. 357-362 (1995). There is no suggestion offollowing the temperature dependence of probe hybridization withfluorescence to identify sequence alterations in PCR products.

The specific hybridization of nucleic acid to a complementary strand foridentification has been exploited in many different formats. Forexample, after restriction enzyme digestion, genomic DNA can be sizefractionated and hybridized to probes by Southern blotting. As anotherexample, single base mutations can be detected by “dot blots” withallele-specific oligonucleotides. Usually, hybridization is performedfor minutes to hours at a single temperature to achieve the necessarydiscrimination. Alternately, the extent of hybridization can bedynamically monitored while the temperature is changing by usingfluorescence techniques. For example, fluorescence melting curves havebeen used to monitor hybridization. L. E. Morrison & L. M. Stols,Sensitive fluorescence-based thermodynamic and kinetic measurements ofDNA hybridization in solution, 32 Biochemistry 3095-3104, 1993). Thetemperature scan rates are usually 10° C./hour or less, partly becauseof the high thermal mass of the fluorimeter cuvette.

Current methods for monitoring hybridization require a lot of time. Ifhybridization could be followed in seconds rather than hours,hybridization could be monitored during PCR amplification, even duringrapid cycle PCR. The many uses of monitoring hybridization during PCR,as will be fully disclosed herein, include, product identification andquantification, sequence alteration detection, and automatic control oftemperature cycling parameters by fluorescence feedback

The prior art, as explained above, carries out temperature cyclingslowly and empirically. When analysis of PCR products by hybridizationis needed, additional time consuming steps are required. Thus, it wouldbe a great advance in the art to provide methods for monitoringhybridization during PCR and analyzing the reaction while it is takingplace, that is, during or immediately after temperature cycling withoutmanipulation of the sample. By monitoring hybridization during PCR, theunderlying principles that allow PCR to work can be followed and used toanalyze and optimize the reaction during amplification.

BRIEF SUMMARY OF THE INVENTION

It is an object of the present invention to provide adouble-strand-specific DNA dye for monitoring product hybridizationduring PCR.

It is another object of the invention to provide a system foridentifying PCR-amplified products by their fluorescence melting curves.

It is also an object of the invention to provide a method for improvingthe sensitivity of PCR quantification with double-strand-specific DNAdyes.

It is still another objection of the invention for determining theamount of specific product amplified by PCR by melting curves to correctfor nonspecific amplification detected with the double-strand-specificDNA dye.

It is a further object of the invention to provide a method of relativequantification of different PCR products with double-strand-specificdyes.

It is yet another object of the invention to provide a method of productquantification by the reannealing kinetics of the product in thepresence of a double-strand-specific DNA dye.

It is a still further object of the invention to provide a novelresonance energy transfer pair to monitor primer and/or probehybridization.

It is still another object of the invention to provide a method ofproduct quantification by the reannealing kinetics of a probe to theproduct using a resonance energy transfer pair.

It is also an object of the present invention to provide a method todetermine initial template copy number by following the fluorescence ofa hybridization probe or probes each cycle during PCR amplification.

It is another object of the invention to provide a system forhomogeneous detection of PCR products by resonance energy transferbetween two labeled probes that hybridize internal to the PCR primers.

It is still another object of the invention to provide a system forhomogeneous detection of PCR products by resonance energy transferbetween one labeled primer and one labeled probe that hybridizesinternal to the PCR primers.

It is yet another object of the invention to provide a system fordetection of sequence alterations internal to PCR primers by resonanceenergy transfer and probe melting curves.

It is a further object of the invention to provide a system for relativequantification of different PCR products by probe melting curves.

It is yet another object of the invention to provide methods todetermine the initial template copy number by curve fitting thefluorescence vs cycle number plot.

It is still another object of the invention to provide a system andmethod for performing PCR rapidly and also continuously monitoring thereaction and adjusting the reaction parameters while the reaction isongoing.

It is another object of the invention to replace the nucleic acid probesby synthetic nucleic acid analogs or derivatives, e.g. by peptidenucleic acids (PNA), provided that they can also be labeled withfluorescent compounds.

These and other objects and advantages of the invention will become morefully apparent from the description and claims which follow, or may belearned by the practice of the invention.

The present invention particularly decreases the total time required forPCR amplification and analysis over prior art techniques while at thesame time allowing the option of significantly increasing the quality ofthe reaction by optimizing amplification conditions.

The present invention provides methods and applications for continuousfluorescence monitoring of DNA amplification. Required instrumentationcombines optical components with structures to provide rapid temperaturecycling to continuously monitor DNA amplification by a variety ofdifferent fluorescence techniques. In one illustrative embodiment,fluorescence is acquired continuously from a single sample oralternately from multiple samples on a rotating carousel with all of thesamples being simultaneously subjected to rapid thermal cycling. Furtherinformation on associated instrumentation can be found in the U.S.patent applications referenced above.

In accordance with one aspect of the present invention, fluorescenceduring DNA amplification was monitored by: 1) the double strand-specificdye SYBR Green I, and 2) resonance energy transfer of fluorescein toCy5™ or Cy5.5™ with hybridization probes. Fluorescence data acquiredonce per cycle allow quantification of initial template copy number.

Furthermore, in contrast to measuring fluorescence once per cycle,embodiments of the present invention are disclosed which monitortemperature, time and fluorescence continuously throughout each cyclethus producing a 3-dimensional spiral. This 3-dimensional spiral can bereduced to temperature vs. time, fluorescence vs. time, and fluorescencevs. temperature plots. Fluorescence vs. temperature plots of thefluorescence from hybridization probes can be used to detect sequencealterations in the product. These sequence alterations may be natural,as in mutations or polymorphisms, or artificial, as in an engineeredalternative template for quantitative PCR.

In accordance with another aspect of the present invention, fluorescencemonitoring is used to acquire product melting curves during PCR byfluorescence monitoring with double-strand-specific DNA specific dyes.Plotting fluorescence as a function of temperature as the thermal cyclerheats through the dissociation temperature of the product gives a PCRproduct melting curve. The shape and position of this DNA melting curveis a function of GC/AT ratio, length, and sequence, and can be used todifferentiate amplification products separated by less than 2° C. inmelting temperature. Desired products can be distinguished fromundesired products, including primer dimers. Analysis of melting curvescan be used to extend the dynamic range of quantitative PCR and todifferentiate different products in multiplex amplification. Usingdouble strand dyes, product denaturation, reannealing and extension canbe followed within each cycle. Continuous monitoring of fluorescenceallows acquisition of melting curves and product annealing curves duringtemperature cycling.

The present invention provides reagents and methods for rapid cycle PCRwith combined amplification and analysis by fluorescence monitoring inunder thirty minutes, more preferably in under fifteen minutes, and mostpreferably in under ten minutes.

A method for analyzing a target DNA sequence of a biological samplecomprises

amplifying the target sequence by polymerase chain reaction in thepresence of two nucleic acid probes that hybridize to adjacent regionsof the target sequence, one of the probes being labeled with an acceptorfluorophore and the other probe labeled with a donor fluorophore of afluorescence energy transfer pair such that upon hybridization of thetwo probes with the target sequence, the donor and acceptor fluorophoresare within 25 nucleotides of one another, the polymerase chain reactioncomprising the steps of adding a thermostable polymerase and primers forthe targeted nucleic acid sequence to the biological sample andthermally cycling the biological sample between at least a denaturationtemperature and an elongation temperature;

exciting the biological sample with light at a wavelength absorbed bythe donor fluorophore and detecting the emission from the fluorescenceenergy transfer pair.

A method for analyzing a target DNA sequence of a biological samplecomprises

amplifying the target sequence by polymerase chain reaction in thepresence of two nucleic acid probes that hybridize to adjacent regionsof the target sequence, one of the probes being labeled with an acceptorfluorophore and the other probe labeled with a donor fluorophore of afluorescence energy transfer pair such that upon hybridization of thetwo probes with the target sequence, the donor and acceptor fluorophoresare within 25 nucleotides of one another, the polymerase chain reactioncomprising the steps of adding a thermostable polymerase and primers forthe targeted nucleic acid sequence to the biological sample andthermally cycling the biological sample between at least a denaturationtemperature and an elongation temperature;

exciting the sample with light at a wavelength absorbed by the donorfluorophore; and

monitoring the temperature dependent fluorescence from the fluorescenceenergy transfer pair.

A method of real time monitoring of a polymerase chain reactionamplification of a target nucleic acid sequence in a biological samplecomprises

(a) adding to the biological sample an effective amount of two nucleicacid primers and a nucleic acid probe, wherein one of the primers andthe probe are each labeled with one member of a fluorescence energytransfer pair comprising an acceptor fluorophore and a donorfluorophore, and wherein the labeled probe hybridizes to an amplifiedcopy of the target nucleic acid sequence within 15 nucleotides of thelabeled primer;

(b) amplifying the target nucleic acid sequence by polymerase chainreaction;

(c) illuminating the biological sample with light of a selectedwavelength that is absorbed by said donor fluorophore; and

(d) detecting the fluorescence emission of the sample.

An improved method of amplifying a target nucleic acid sequence of abiological sample comprises

(a) adding to the biological sample an effective amount of anucleic-acid-binding fluorescent entity;

(b) amplifying the target nucleic acid sequence using polymerase chainreaction, comprising thermally cycling the biological sample usinginitial predetermined temperature and time parameters, and then

(i) illuminating the biological sample with a selected wavelength oflight that is absorbed by the fluorescent entity during the polymerasechain reaction;

(ii) monitoring fluorescence from the sample to determine the optimaltemperature and time parameters for the polymerase chain reaction; and

(iii) adjusting the initial temperature and time parameters inaccordance with the fluorescence.

In one illustrative embodiment, the fluorescent entity comprises adouble strand specific nucleic acid binding dye, and in anotherillustrative embodiment the fluorescent entity comprises a fluorescentlylabeled oligonucleotide probe that hybridizes to the targeted nucleicacid sequence.

A method for detecting a target nucleic acid sequence of a biologicalsample comprises

(a) adding to the biological sample an effective amount of a pair ofoligonucleotide probes that hybridize to the target nucleic acidsequence, one of the probes being labeled with an acceptor fluorophoreand the other probe labeled with a donor fluorophore of a fluorescenceenergy transfer pair, wherein an emission spectrum of the donorfluorophore and an absorption spectrum of the acceptor fluorophoreoverlap less than 25%, the acceptor fluorophore has a peak extinctioncoefficient greater than 100,000 M⁻¹ cm⁻¹ and upon hybridization of thetwo probes, the donor and acceptor fluorophores are within 25nucleotides of one another;

(b) illuminating the biological sample with a selected wavelength oflight that is absorbed by said donor fluorophore; and

(c) detecting the emission of the biological sample. An illustrativeresonance energy transfer pair comprises fluorescein as the donor andCy5 or Cy5.5 as the acceptor.

A method of real time monitoring of a polymerase chain reactionamplification of a target nucleic acid sequence in a biological samplecomprises

amplifying the target sequence by polymerase chain reaction in thepresence of two nucleic acid probes that hybridize to adjacent regionsof the target sequence, one of the probes being labeled with an acceptorfluorophore and the other probe labeled with a donor fluorophore of afluorescence energy transfer pair such that upon hybridization of thetwo probes with the target sequence, the donor and acceptor fluorophoresare within 25 nucleotides of one another, the polymerase chain reactioncomprising the steps of adding a thermostable polymerase and primers forthe targeted nucleic acid sequence to the biological sample andthermally cycling the biological sample between at least a denaturationtemperature and an elongation temperature;

exciting the biological sample with light at a wavelength absorbed bythe donor fluorophore and detecting the emission from the biologicalsample; and

monitoring the temperature dependent fluorescence from the fluorescenceenergy transfer pair.

A method of real time monitoring of a polymerase chain reactionamplification of a target nucleic acid sequence in a biological samplecomprises

amplifying the target sequence by polymerase chain reaction in thepresence of SYBR™ Green I, the polymerase chain reaction comprising thesteps of adding a thermostable polymerase and primers for the targetednucleic acid sequence to the biological sample and thermally cycling thebiological sample between at least a denaturation temperature and anelongation temperature;

exciting the biological sample with light at a wavelength absorbed bythe SYBR™ Green I and detecting the emission from the biological sample;and

monitoring the temperature dependent fluorescence from the SYBR™ GreenI. Preferably, the monitoring step comprises determining a meltingprofile of the amplified target sequence.

A method for analyzing a target DNA sequence of a biological samplecomprises

(a) adding to the biological sample an effective amount of two nucleicacid primers and a nucleic acid probe, wherein one of the primers andthe probe are each labeled with one member of a fluorescence energytransfer pair comprising an acceptor fluorophore and a donorfluorophore, and wherein the labeled probe hybridizes to an amplifiedcopy of the target nucleic acid sequence within 15 nucleotides of thelabeled primer;

(b) amplifying the target nucleic acid sequence by polymerase chainreaction;

(c) illuminating the biological sample with light of a selectedwavelength that is absorbed by said donor fluorophore and detecting thefluorescence emission of the sample. In another illustrative embodiment,the method further comprises the step of monitoring the temperaturedependent fluorescence of the sample, preferably by determining amelting profile of the amplified target sequence.

A method of detecting a difference at a selected locus in a firstnucleic acid as compared to a second nucleic acid comprises

(a) providing a pair of oligonucleotide primers configured foramplifying, by polymerase chain reaction, a selected segment of thefirst nucleic acid and a corresponding segment of the second nucleicacid, wherein the selected segment and corresponding segment eachcomprises the selected locus, to result in amplified products containinga copy of the selected locus;

(b) providing a pair of oligonucleotide probes, one of the probes beinglabeled with an acceptor fluorophore and the other probe being labeledwith a donor fluorophore of a fluorogenic resonance energy transfer pairsuch that upon hybridization of the two probes with the amplifiedproducts the donor and acceptor are in resonance energy transferrelationship, wherein one of the probes is configured for hybridizing tothe amplified products such that said one of the probes spans theselected locus and exhibits a melting profile when the difference ispresent in the first nucleic acid that is distinguishable from a meltingprofile of the second nucleic acid;

(c) amplifying the selected segment of first nucleic acid and thecorresponding segment of the second nucleic acid by polymerase chainreaction in the presence of effective amounts of probes to result in anamplified selected segment and an amplified corresponding segment, atleast a portion thereof having both the probes hybridized thereto withthe fluorogenic resonance energy transfer pair in resonance energytransfer relationship;

(d) illuminating the amplified selected segment and the amplifiedcorresponding segment with the probes hybridized thereto with a selectedwavelength of light to elicit fluorescence by the fluorogenic resonanceenergy transfer pair;

(e) measuring fluorescence emission as a function of temperature todetermine in a first melting profile of said one of the probes meltingfrom the amplified selected segment of first nucleic acid and a secondmelting profile of said one of the probes melting from the amplifiedcorresponding segment of second nucleic acid; and

(f) comparing the first melting profile to the second melting profile,wherein a difference therein indicates the presence of the difference inthe sample nucleic acid.

A method of detecting a difference at a selected locus in a firstnucleic acid as compared to a second nucleic acid comprises

(a) providing a pair of oligonucleotide primers configured foramplifying, by polymerase chain reaction, a selected segment of thefirst nucleic acid and a corresponding segment of the second nucleicacid, wherein the selected segment and corresponding segment eachcomprises the selected locus, to result in amplified products containinga copy of the selected locus;

(b) providing an oligonucleotide probe, wherein one of the primers andthe probe are each labeled with one member of a fluorescence energytransfer pair comprising an donor fluorophore and an acceptorfluorophore, and wherein the labeled probe and labeled primer hybridizeto the amplified products such that the donor and acceptor are inresonance energy transfer relationship, and wherein the probe isconfigured for hybridizing to the amplified products such that saidprobe spans the selected locus and exhibits a melting profile when thedifference is present in the first nucleic acid that is distinguishablefrom a melting profile of the second nucleic acid;

(c) amplifying the selected segment of first nucleic acid and thecorresponding segment of the second nucleic acid by polymerase chainreaction in the presence of effective amounts of primers and probe toresult in an amplified selected segment and an amplified correspondingsegment, at least a portion thereof having the labled primer and probehybridized thereto with the fluorogenic resonance energy transfer pairin resonance energy transfer relationship;

(d) illuminating the amplified selected segment and the amplifiedcorresponding segment with the labeled primer and probe hybridizedthereto with a selected wavelength of light to elicit fluorescence bythe fluorogenic resonance energy transfer pair;

(e) measuring fluorescence emission as a function of temperature todetermine in a first melting profile of said probe melting from theamplified selected segment of first nucleic acid and a second meltingprofile of said probe melting from the amplified corresponding segmentof second nucleic acid; and

(f) comparing the first melting profile to the second melting profile,wherein a difference therein indicates the presence of the difference inthe sample nucleic acid.

A method of detecting heterozygosity at a selected locus in the genomeof an individual, wherein the genome comprises a mutant allele and acorresponding reference allele, each comprising the selected locus,comprises

(a) obtaining sample genomic DNA from the individual;

(b) providing a pair of oligonucleotide primers configured foramplifying, by polymerase chain reaction, a first selected segment ofthe mutant allele and a second selected segment of the correspondingreference allele wherein both the first and second selected segmentscomprise the selected locus;

(c) providing a pair of oligonucleotide probes, one of the probes beinglabeled with an acceptor fluorophore and the other probe being labeledwith a donor fluorophore of a fluorogenic resonance energy transfer pairsuch that upon hybridization of the two probes with the amplified firstand second selected segments one of the probes spans the selected locusand exhibits a first melting profile with the amplified first selectedsegment that is distinguishable from a second melting profile with theamplified second selected segment;

(d) amplifying the first and second selected segments of sample genomicDNA by polymerase chain reaction in the presence of effective amounts ofprobes to result in amplified first and second selected segments, atleast a portion thereof having both the probes hybridized thereto withthe fluorogenic resonance energy transfer pair in resonance energytransfer relationship;

(e) illuminating the amplified first and second selected segments havingthe probes hybridized thereto with a selected wavelength of light toelicit fluorescence by the donor and acceptor;

(f) measuring a fluorescence emission as a function of temperature todetermine a first melting profile of said one of the probes melting fromthe amplified first selected segment and a second melting profile ofsaid one of the probes melting from the amplified second selectedsegment; and

(g) comparing the first melting profile to the second melting profile,wherein distinguishable melting profiles indicate heterozygosity in thesample genomic DNA.

A method of detecting heterozygosity at a selected locus in the genomeof an individual, wherein the genome comprises a mutant allele and acorresponding reference allele, each comprising the selected locus,comprises

(a) obtaining sample genomic DNA from the individual;

(b) providing a pair of oligonucleotide primers configured foramplifying, by polymerase chain reaction, a first selected segment ofthe mutant allele and a second selected segment of the correspondingreference allele wherein both the first and second selected segmentscomprise the selected locus;

(c) providing an oligonucleotide probe, wherein one of the primers andthe probe are each labeled with one member of a fluorescence energytransfer pair comprising an donor fluorophore and an acceptorfluorophore, and wherein the labeled probe and labeled primer hybridizeto the amplified first and second selected segments such that one of theprobes spans the selected locus and exhibits a first melting profilewith the amplified first selected segment that is distinguishable from asecond melting profile with the amplified second selected segment;

(d) amplifying the first and second selected segments of sample genomicDNA by polymerase chain reaction in the presence of effective amounts ofprimers and probe to result in amplified first and second selectedsegments, at least a portion thereof having both the labeled primer andprobe hybridized thereto with the fluorogenic resonance energy transferpair in resonance energy transfer relationship;

(e) illuminating the amplified first and second selected segments havingthe labeled primer and probe hybridized thereto with a selectedwavelength of light to elicit fluorescence by the donor and acceptor;

(f) measuring a fluorescence emission as a function of temperature todetermine a first melting profile of said probe melting from theamplified first selected segment and a second melting profile of saidprobe melting from the amplified second selected segment; and

(g) comparing the first melting profile to the second melting profile,wherein distinguishable melting profiles indicate heterozygosity in thesample genomic DNA.

A method of determining completion of a polymerase chain reaction in apolymerase chain reaction mixture comprising (1) a nucleic acid whereinthe nucleic acid or a polymerase-chain-reaction-amplified productthereof consists of two distinct complementary strands, (2) twooligonucleotide primers configured for amplifying by polymerase chainreaction a selected segment of the nucleic acid to result in anamplified product, and (3) a DNA polymerase for catalyzing thepolymerase chain reaction, comprises

(a) adding to the mixture (1) an effective amount of an oligonucleotideprobe labeled with a resonance energy transfer donor or a resonanceenergy transfer acceptor of a fluorogenic resonance energy transferpair, wherein the probe is configured for hybridizing to the amplifiedproduct under selected conditions of temperature and monovalent ionicstrength, and (2) an effective amount of a reference oligonucleotidelabeled with the donor or the acceptor, with the proviso that as betweenthe probe and reference oligonucleotide one is labeled with the donorand the other is labeled with the acceptor, wherein the referenceoligonucleotide is configured for hybridizing to the amplified productunder the selected conditions of temperature and monovalent ionicstrength such that the donor and the acceptor are in resonance energytransfer relationship when both the probe and the referenceoligonucleotide hybridize to the amplified product;

(b) amplifying the selected segment of nucleic acid by polymerase chainreaction to result in the amplified product, at least a portion thereofhaving both the probe and the reference oligonucleotide hybridizedthereto with the fluorogenic resonance energy transfer pair in resonanceenergy transfer relationship; and

(c) illuminating the amplified product having the probe and referenceoligonucleotide hybridized thereto with a selected wavelength of lightfor eliciting fluorescence by the fluorogenic resonance energy pair andmonitoring fluorescence emission and determining a cycle when thefluorescence emission reaches a plateau phase, indicating the completionof the reaction.

A method of determining completion of a polymerase chain reaction in apolymerase chain reaction mixture comprising (1) a nucleic acid whereinthe nucleic acid or a polymerase-chain-reaction-amplified productthereof consists of two distinct complementary strands, (2) twooligonucleotide primers configured for amplifying by polymerase chainreaction a selected segment of the nucleic acid to result in anamplified product, and (3) a DNA polymerase for catalyzing thepolymerase chain reaction, comprises

(a) adding to the mixture an effective amount of a nucleic-acid-bindingfluorescent dye;

(b) amplifying the selected segment of nucleic acid by polymerase chainreaction in the mixture to which the nucleic-acid-binding fluorescentdye has been added to result in the amplified product withnucleic-acid-binding fluorescent dye bound thereto; and

(c) illuminating amplified product with nucleic-acid-binding fluorescentdye bound thereto with a selected wavelength of light for elicitingfluorescence therefrom and monitoring fluorescence emission anddetermining a cycle when the fluorescence emission reaches a plateauphase, indicating the completion of the reaction. Preferably, thenucleic-acid-binding fluorescent dye is a member selected from the groupconsisting of SYBR™ GREEN I, ethidium bromide, pico green, acridineorange, thiazole orange, YO-PRO-1, and chromomycin A3, and morepreferably is SYBR™ GREEN I.

A method of controlling temperature cycling parameters of a polymerasechain reaction comprising repeated cycles of annealing, extension, anddenaturation phases of a polymerase chain reaction mixture comprising adouble-strand-specific fluorescent dye, wherein the parameters compriseduration of the annealing phase, duration of the denaturation phase, andnumber of cycles, comprises

(a) illuminating the reaction with a selected wavelength of light foreliciting fluorescence from the fluorescent dye and continuouslymonitoring fluorescence during the repeated annealing, extension, anddenaturation phases;

(b) determining at least

(i) duration for fluorescence to stop increasing during the extensionphase, or

(ii) Duration for fluorescence to decrease to a baseline level duringthe denaturation phase, or

(iii) a number of cycles for fluorescence to reach a preselected levelduring the extension phase; and

(c) adjusting the length of the extension phase according to the lengthof time for fluorescence to stop increasing during the extension phase,the length of the denaturation phase according to the length of time forfluorescence to decrease to the baseline level during the denaturationphase, or the number of cycles according to the number of cycles forfluorescence to reach the preselected level during the extension phase.

A method of determining a concentration of an amplified product in aselected polymerase chain reaction mixture comprises

(a) determining a second order rate constant for the amplified productat a selected temperature and reaction conditions by monitoring rate ofhybridization of a known concentration of the amplified product;

(b) determining rate of annealing for an unknown concentration of theamplified product; and

(c) calculating the concentration of the amplified product from the rateof annealing and the second order rate constant.

Preferably, the rate of annealing is determined after multiple cycles ofamplification. One illustrative method of determining the second orderrate constant comprises the steps of

raising the temperature of a first polymerase chain reaction mixturecomprising a known concentration of the amplified product and aneffective amount of a double-strand specific fluorescent dye, above thedenaturation temperature of the amplified product to result in adenatured amplified product;

rapidly reducing the temperature of the first polymerase chain reactionmixture comprising the known amount of denatured amplified product to aselected temperature below the denaturation temperature of the amplifiedproduct while continuously monitoring the fluorescence of the firstpolymerase chain reaction mixture as a function of time;

plotting fluorescence as a function of time for determining maximumfluorescence, minimum fluorescence, the time at minimum fluorescence,and a second order rate constant for the known concentration ofamplified product from the equation$F = {F_{\max} - \frac{F_{\max} - F_{\min}}{{{k\left( {t - t_{0}} \right)}\lbrack{DNA}\rbrack} + 1}}$

wherein F is fluorescence, F_(max) is maximum fluorescence, F_(min) isminimum fluorescence, k is the second order rate constant, t₀ is thetime at F_(min), and [DNA] is the known concentration of the amplifiedproduct.

A method of determining a concentration of a selected nucleic acidtemplate by competitive quantitative polymerase chain reaction comprisesthe steps of:

(a) in a reaction mixture comprising:

(i) effective amounts of each of a pair of oligonucleotide primersconfigured for amplifying, in a polymerase chain reaction, a selectedsegment of the selected template and a corresponding selected segment ofa competitive template to result in amplified products thereof,

(ii) an effective amount of an oligonucleotide probe labeled with aresonance energy transfer donor or a resonance energy transfer acceptorof a fluorogenic resonance energy transfer pair, wherein the probe isconfigured for hybridizing to the amplified products such that the probemelts from the amplified product of the selected template at a meltingtemperature that is distinguishable from the melting temperature atwhich the probe melts from the amplified product of the competitivetemplate,

(iii) an effective amount of a reference oligonucleotide labeled withthe donor or the acceptor, with the proviso that as between the probeand transfer oligonucleotide one is labeled with the donor and the otheris labeled with the acceptor, wherein the reference oligonucleotide isconfigured for hybridizing to the amplified products such that the donorand the acceptor are in resonance energy transfer relationship when boththe probe and the

reference oligonucleotide hybridize to the amplified products;amplifying, by polymerase chain reaction, an unknown amount of theselected template and a known amount of the competitive template toresult in the amplified products thereof;

(b) illuminating the reaction mixture with a selected wavelength oflight for eliciting fluorescence by the fluorogenic resonance energytransfer pair and determining a fluorescence emission as a function oftemperature as the temperature of the reaction mixture is changed toresult in a first melting curve of the probe melting from the amplifiedproduct of the selected template and a second melting curve of the probemelting from the competitive template;

(c) converting the first and second melting curves to first and secondmelting melting peaks and determining relative amounts of the selectedtemplate and the competitive template from such melting peaks; and

(d) calculating the concentration of the selected template based on theknown amount of the competitive template and the relative amounts ofselected template and competitive template.

A fluorogenic resonance energy transfer pair consists of fluorescein andCy5 or Cy5.5.

A method of determining a concentration of a selected nucleic acidtemplate in a polymerase chain reaction comprises the steps of:

(a) in a reaction mixture comprising:

(i) effective amounts of each of a first pair of oligonucleotide primersconfigured for amplifying, in a polymerase chain reaction, a selectedfirst segment of the selected template to result in an amplified firstproduct thereof,

(ii) effective amounts of each of a second pair of oligonucleotideprimers configured for amplifying, in a polymerase chain reaction, aselected second segment of a reference template to result in anamplified second product thereof,

(iii) an effective amount of a nucleic-acid-binding fluorescent dye;amplifying, by polymerase chain reaction, an unknown amount of theselected template to result in the amplified first product and a knownamount of the reference template to result in the amplified secondproduct thereof;

(b) illuminating the reaction mixture with a selected wavelength oflight for eliciting fluorescence by the nucleic-acid-binding fluorescentdye and continuously monitoring the fluorescence emitted as a functionof temperature to result in a melting curve of the amplified productswherein the first product and second product melt at differenttemperatures;

(c) converting the melting curves to melting melting peaks anddetermining relative amounts of the selected template and the referencetemplate from such melting peaks; and

(d) calculating the concentration of the selected template based on theknown amount of the reference template and the relative amounts ofselected template and reference template.

A method of monitoring amplification of a selected template in apolymerase chain reaction that also comprises a positive controltemplate comprises the steps of:

(a) in a reaction mixture comprising:

(i) effective amounts of each of a first pair of oligonucleotide primersconfigured for amplifying, in a polymerase chain reaction, a selectedfirst segment of the selected template to result in an amplified firstproduct thereof,

(ii) effective amounts of each of a second pair of oligonucleotideprimers configured for amplifying, in a polymerase chain reaction, aselected second segment of the positive control template to result in anamplified second product thereof,

(iii) an effective amount of a nucleic-acid-binding fluorescent dye;subjecting the selected template and the positive control template toconditions for amplifying the selected template and the positive controltemplate by polymerase chain reaction; and

(b) illuminating the reaction mixture with a selected wavelength oflight for eliciting fluorescence by the nucleic-acid-binding fluorescentdye and continuously monitoring the fluorescence emitted as a functionof temperature during an amplification cycle of the polymerase chainreaction to result in a first melting peak of the amplified firstproduct, if the selected template is amplified, and a second meltingpeak of the amplified second product, if the positive control templateis amplified;

wherein obtaining of the second melting curve indicates that thepolymerase chain reaction was operative, obtaining the first meltingcurve indicates that the selected first segment was amplifiable, andabsence of the first melting curve indicates that the selected firstsegment was not amplifiable.

A method of detecting the factor V Leiden mutation in an individual,wherein the factor V Leiden mutation consists of a single base change atthe factor V Leiden mutation locus as compared to wild type, comprisesthe steps of:

(a) obtaining sample genomic DNA from the individual;

(b) providing wild type genomic DNA as a control;

(c) providing a pair of oligonucleotide primers configured foramplifying by polymerase chain reaction a selected segment of the samplegenomic DNA and of the wild type genomic DNA wherein the selectedsegment comprises the factor V Leiden mutation locus to result inamplified products containing a copy of the factor V Leiden mutationlocus;

(d) providing an oligonucleotide probe labeled with a resonance energytransfer donor or a resonance energy transfer acceptor of a fluorogenicresonance energy transfer pair, wherein the probe is configured forhybridizing to the amplified products such that the probe spans themutation locus and exhibits a melting profile when the factor V Leidenmutation is present in the sample genomic DNA that is differentiablefrom a melting profile of the wild type genomic DNA;

(e) providing a transfer oligonucleotide labeled with the resonanceenergy transfer donor or the resonance energy transfer acceptor, withthe proviso that as between the probe and transfer oligonucleotide oneis labeled with the resonance energy transfer donor and the other islabeled with the resonance energy transfer acceptor, wherein thetransfer oligonucleotide is configured for hybridizing to the amplifiedproducts such that the resonance energy transfer donor and the resonanceenergy transfer acceptor are in resonance energy transfer relationshipwhen both the probe and the transfer oligonucleotide hybridize to theamplified products;

(f) amplifying the selected segment of sample genomic DNA and wild typegenomic DNA by polymerase chain reaction in the presence of effectiveamounts of oligonucleotide probe and transfer oligonucleotide to resultin amplified selected segments, at least a portion thereof having boththe probe and the transfer oligonucleotide hybridized thereto with thefluorogenic resonance energy transfer pair in resonance energy transferrelationship;

(g) determining fluorescence as a function of temperature during anamplification cycle of the polymerase chain reaction to result in amelting profile of the probe melting from the amplified segment ofsample genomic DNA and a melting profile of the probe melting from theamplified segment of wild type genomic DNA; and

(h) comparing the melting profile for the sample genomic DNA to themelting profile for the wild type genomic DNA, wherein a differencetherein indicates the presence of the factor V Leiden mutation in thesample genomic DNA.

A method of analyzing nucleic acid hybridization comprises the steps of

(a) providing a mixture comprising a nucleic acid sample to be analyzedand a nucleic acid binding fluorescent entity; and

(b) monitoring fluorescence while changing temperature at a rate of≧0.1° C./second.

A method of quantitating an initial copy number of a sample containingan unknown amount of nucleic acid comprises the steps of

(a) amplifying by polymerase chain reaction at least one standard ofknown concentration in a mixture comprising the standard and a nucleicacid binding fluorescent entity;

(b) measuring fluorescence as a function of cycle number to result in aset of data points;

(c) fitting the data points to a given predetermined equation describingfluorescence as a function of initial nucleic acid concentration andcycle number;

(d) amplifying the sample containing the unknown amount of nucleic acidin a mixture comprising the sample and the nucleic acid bindingfluorescent entity and monitoring fluorescence thereof; and

(e) determining initial nucleic acid concentration from the equationdetermined in step (c).

A fluorescence resonance energy transfer pair is disclosed wherein thepair comprises a donor fluorophore having an emission spectrum and anacceptor fluorophore having an absorption spectrum and an extinctioncoefficient greater than 100,000 M⁻¹ cm⁻¹, wherein the donorfluorophore's emission spectrum and the acceptor fluorophore'sabsorption spectrum overlap less than 25%. One illustrative fluorescenceresonance energy transfer pair described is where the donor fluorophoreis fluorescein and the acceptor fluorophore is Cy5 or Cy5.5.

A method for analyzing a target DNA sequence of a biological samplecomprises

amplifying the target sequence by polymerase chain reaction in thepresence of a nucleic acid binding fluorescent entity, said polymerasechain reaction comprising the steps of adding a thermostable polymeraseand primers for the targeted nucleic acid sequence to the biologicalsample and thermally cycling the biological sample between at least adenaturation temperature and an elongation temperature;

exciting the sample with light at a wavelength absorbed by the nucleicacid binding fluorescent entity; and

monitoring the temperature dependent fluorescence from the nucleic acidbinding fluorescent entity as temperature of the sample is changed.Preferably, the nucleic acid binding fluorescent entity comprises adouble stranded nucleic acid binding fluorescent dye, such as SYBR™Green I. The temperature dependent fluorescence can be used to identifythe amplified products, preferably by melting curve analysis. Relativeamounts fo two or more amplified products can be determined by analysisof melting curves. For example, areas under the melting curves are foundby non-linear least squares regression of the sum of multiple gaussians.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

FIGS. 1A&B are graphs representing an equilibrium PCR paradigm (A) and akinetic PCR paradigm (B).

FIG. 2 illustrates useful temperature v. time segments for fluorescencehybridization monitoring.

FIG. 3 is a temperature v. time chart exemplary of rapid temperaturecycling for PCR.

FIG. 4 shows the results of four different temperature/time profiles(A-D) and their resultant amplification products after thirty cycles(inset).

FIGS. 5A, B & C illustrate the mechanism of fluorescence generation forthree different methods of fluorescence monitoring of PCR: (A)(1) and(A)(2) double-stranded DNA dye, (B)(1) and (B)(2) hydrolysis probe, and(C)(1) and (C)(2) hybridization probes.

FIG. 6 shows the chemical structure of the monovalentN-hydroxysuccinimide ester of Cy5™.

FIG. 7 shows the chemical structure of the monovalentN-hydroxysuccinimide ester of Cy5.5™.

FIG. 8 shows the emission spectrum of fluorescein (solid line) and theexcitation spectrum of Cy5 (broken line).

FIG. 9 shows resonance energy transfer occurring between fluorescein-and Cy5-labeled adjacent hybridization probes at each cycle during PCR.

FIG. 10 shows the effect of varying the ratio of the Cy5 probe to thefluorescein probe on the resonance energy transfer signal generatedduring PCR.

FIG. 11 shows the effect of varying the probe concentration at a givenprobe ratio on the resonance energy transfer signal generated duringPCR.

FIG. 12 shows the effect of spacing between the labeled oligonucleotideson the resonance energy transfer signal generated during PCR.

FIG. 13 shows the time course of adjacent probe hybridization byfluorescence energy transfer immediately after 30 cycles ofamplification with Taq DNA polymerase (exo⁺; solid line) and the Stoffelfragment of Taq DNA polymerase (exo⁻; dotted line) of temperaturecycling and the type of polymerase on fluorescence development duringPCR with adjacent hybridization probes; temperature is shown with a boldline.

FIG. 14 is a fluorescence ratio v. cycle number plot for amplificationwith Taq DNA polymerase (exo⁻; solid line), Stoffel fragment of Taq DNApolymerase (exo⁻; broken line), and KlenTaq DNA polymerase (exo⁻; dottedline): top panel-cycling is between 94° C. and 60° C. with a 20 secondhold at 60° C.; middle panel-cycling is between 94° C. and 60° C. with a120 second hold at 60° C.; bottom panel-cycling is between 94° C. and60° C. with a slow increase from 60° C. to 75° C.

FIG. 15 is a fluorescence v. cycle number plot for a number of differentinitial template copy reactions monitored with Sybr™ Green I: 0, (Δ); 1,(▪); 10, (−); 10², (−); 10³, (+); 10⁴, (); 10⁵, (⋄); 10⁶, (×); 10⁷,(Δ); 10⁸, (□); 10⁹, (♦).

FIG. 16 is a fluorescence ratio v. cycle number plot for a number ofdifferent initial template copy reactions monitored with a dual-labeledhydrolysis probe: 0, (−); 1, (▴); 10, (◯); 10², (*); 10³, (); 10⁴, (□);10⁵, (+); 10⁶, (▪); 10⁷, (⋄); 10 ⁸, (×); 10⁹, (♦).

FIG. 17 is a fluorescence ratio v. cycle number plot for a number ofdifferent initial template copy reactions monitored with adjacenthybridization probes: 0, (−); 1, (▴); 10, (◯); 10², (*); 10³, (); 10⁴,(□); 10⁵, (+); 10⁶, (▪); 10⁷, (⋄); 10⁸, (×); 10⁹, (♦).

FIG. 18 is a fluorescence ratio v. cycle number plot distinguishing twohybridization probe designs monitored by resonance energy transfer: (⋄)two hybridization probes labeled respectively with fluorescein and Cy5;and (♦) a primer labeled with Cy5 and a probe labeled with fluorescein.

FIGS. 19A-C provide a comparison of three fluorescence monitoringtechniques for PCR, including the double-strand specific DNA dye SYBRGreen I (A), a dual-labeled fluorescein/rhodamine hydrolysis probe (B),and a fluorescein-labeled hybridization probe with a Cy5-labeled primer(C); FIG. 19D shows the coefficient of variation for the threemonitoring techniques represented in FIGS. 19A-C.

FIG. 20 shows a typical log fluorescence vs cycle number plot of astandard amplification monitored with SYBR Green I.

FIG. 21 shows an expontial curve fit to cycles 20-27 of the data fromFIG. 20.

FIG. 22 shows an exponential fit to an unknown to determine initial copynumber from amplification data.

FIG. 23 shows a typical fluorescence v. cycle number plot of fivestandards monitored each cycle with adjacent hybridization probes,wherein initial copy numbers are represented as follows: 10³, (); 10⁴,(⋄); 10⁵, (▴); 10⁶, (□); 10⁷, (♦).

FIG. 24 shows a curve fit to the standard data of FIG. 23.

FIG. 25 shows a typical fluorescence vs cycle number plot of fivestandards monitored each cycle with a hydrolysis probe, wherein initialcopy numbers are represented as follows: 1.5, (); 15, (⋄); 150, (▴);1500, (□); 15,000, (♦).

FIG. 26 shows a curve fit to the standard data of FIG. 25.

FIG. 27 shows a typical log fluorescence vs cycle number plot of threestandard amplifications monitored with SYBR Green I, wherein: (▪); ();(▴).

FIG. 28 shows different curve fit to the standard data of FIG. 27.

FIGS. 29A&B show plots of (A) time v. fluorescence and (B) time v.temperature demonstrating the inverse relationship between temperatureand fluorescence.

FIG. 30 is a chart showing 2D plots of temperature v. time, fluorescencev. time, and fluorescence v. temperature, also shown as a 3D plot, forthe amplification of a 180 base pair fragment of the hepatitis B genomein the presence of SYBR Green I.

FIG. 31 is a fluorescence v. temperature projection for theamplification of a 536 base pair fragment of the human beta-globin genein the presence of SYBR Green I.

FIGS. 32A&B provide a plot showing (A) a linear change in fluorescenceratio with temperature for hydrolysis probes, and (B) a radical changewith temperature for hybridization probes.

FIG. 33 shows a fluorescence ratio v. temperature plot of amplificationwith an exo⁻ polymerase in the presence of adjacent hybridizationprobes.

FIG. 34 shows a fluorescence ratio v. temperature plot of amplificationwith an exo⁺ polymerase in the presence of adjacent hybridizationprobes.

FIG. 35 shows a 3-dimensional plot of temperature, time and fluorescenceduring amplification with an exo⁻ polymerase in the presence of adjacenthybridization probes.

FIG. 36 shows a 3-dimensional plot of temperature, time, andfluorescence during amplification with an exo⁺ polymerase in thepresence of adjacent hybridization probes.

FIG. 37 shows melting curves for PCR-amplified products of hepatitis Bvirus (; 50% GC, 180 bp); beta-globin (▴; 53.2% GC, 536 bp); andprostate specific antigen (X; 60.3% GC, 292 bp).

FIG. 38 shows melting curves for PCR-amplified product of hepatitis Bvirus at heating rates of 0.1° C. to 5.0° C.

FIG. 39 shows melting curves for PCR-amplified product of hepatitis Bvirus at various SYBR™ Green I concentrations.

FIGS. 40A&B show (A) melting curves and (B) electrophoreticallyfractionated bands of products of a beta-globin fragment amplified with(a) no added template, (b) 10⁶ copies of added template under lowstringency conditions, and (c) 10⁶ copies of added template under higherstringency conditions.

FIGS. 41A&B show (A) melting curves and (B) melting peaks of hepatitis Bvirus fragment (HBV), β-globin, and a mixture thereof.

FIGS. 42A-D show (A) a relative fluorescence v. cycle number plot forPCR amplified products from various amounts of β-globin template, (B)melting peaks and (C) electrophoretic bands of the various products, and(D) corrected fluorescence of the data of (A).

FIGS. 43A&B show (A) melting curves and (B) melting peaks from PCRamplified products of a mixture of the cystic fibrosis gene and thec-erbB-2 oncogene.

FIG. 44 show melting peaks at various cycle numbers for the cysticfibrosis gene (CFTR) and c-erbB-2 (neu).

FIG. 45 shows a graph of integrated melting peaks of CFTR and neu PCTproducts.

FIGS. 46A&B show (A) melting curves and (B) melting peaks for PCRproducts of a person heterozygous for the factor V Leiden mutation(solid line), homozygous for the factor V Leiden mutation (dotted line),homozygous wild type (broken line), and no DNA control (alternating dotand dash).

FIG. 47 shows a fluorescence ratio v. temperature plot of continuousmonitoring during cycle 40 of PCR products of a sample homozygous forthe factor V Leiden mutation (solid line), heterozygous for the factor VLeiden mutation (dotted line), and homozygous wild type (alternating dotand dash).

FIG. 48 shows melting peaks of a homozygous mutant of themethylenetatrahydrofolate gene (solid line), homozygous wild type(broken line), heterozygous mutant (dotted line), and no DNA control(alternating dot and dash).

FIG. 49 shows the shape of reannealing curves of amplified β-globin PCRproducts from various initial template amounts.

FIG. 50 shows the determination of a second order rate constant fordetermining initial template concentration.

FIG. 51 shows a block diagram for controlling thermal cycling fromfluorescence data.

FIGS. 52A&B show (A) a temperature v. time plot acquired after 20 cyclesand (B) a fluorescence v. time plot acquired after 25 cycles whereinthermal cycling was controlled from fluorescence data.

DETAILED DESCRIPTION

Before the present methods for monitoring hybridization during PCR aredisclosed and described, it is to be understood that this invention isnot limited to the particular configurations, process steps, andmaterials disclosed herein as such configurations, process steps, andmaterials may vary somewhat. It is also to be understood that theterminology employed herein is used for the purpose of describingparticular embodiments only and is not intended to be limiting since thescope of the present invention will be limited only by the appendedclaims and equivalents thereof.

It must be noted that, as used in this specification and the appendedclaims, the singular forms “a,” “an,” and “the” include plural referentsunless the context clearly dictates otherwise.

In describing and claiming the present invention, the followingterminology will be used in accordance with the definitions set outbelow.

As used herein, “nucleic acid,” “DNA,” and similar terms also includenucleic acid analogs, i.e. analogs having other than a phosphodiesterbackbone. For example, the so-called “peptide nucleic acids,” which areknown in the art and have peptide bonds instead of phosphodiester bondsin the backbone, are considered within the scope of the presentinvention.

As used herein, “continuous monitoring” and similar terms refer tomonitoring multiple times during a cycle of PCR, preferably duringtemperature transitions, and more preferably obtaining at least one datapoint in each temperature transition.

As used herein, “cycle-by-cycle” monitoring means monitoring the PCRreaction once each cycle, preferably during the annealing phase of PCR.

As used herein, “fluorescence resonance energy transfer relationship”and similar terms refer to adjacent hybridization of an oligonucleotidelabeled with a donor fluorophore and another oligonucleotide labeledwith an acceptor fluorophore to a target nucleic acid such that thedonor fluorophore can transfer resonance energy to the acceptorfluorophore such that the acceptor fluorophore produces a measurablefluorescence emission. If the donor fluorophore and acceptor fluorophoreare spaced apart by too great a distance, then the donor fluorophorecannot transfer resonance energy to the acceptor fluorophore such thatthe acceptor fluorophore emits measurable fluorescence, and hence thedonor fluorophore and acceptor fluorophore are not in resonance energytransfer relationship. Preferably, when the two labeled oligonucleotidesare both probes and neither functions as a PCR primer (“probe-probe”),then the donor and acceptor fluorophores are within about 0-25nucleotides, more preferably within about 0-5 nucleotides, and mostpreferably within about 0-2 nucleotides. A particularly preferredspacing is 1 nucleotide. When one of the labeled oligonucleotides alsofunctions as a PCR primer (“probe-primer”), then the donor and acceptorfluorophores are preferably within about 0-15 nucleotides and morepreferably within about 4-6 nucleotides.

As used herein, “effective amount” means an amount sufficient to producea selected effect. For example, an effective amount of PCR primers is anamount sufficient to amplify a segment of nucleic acid by PCR providedthat a DNA polymerase, buffer, template, and other conditions, includingtemperature conditions, known in the art to be necessary for practicingPCR are also provided.

PCR requires repetitive template denaturation and primer annealing.These hybridization transitions are temperature-dependent. Thetemperature cycles of PCR that drive amplification alternately denatureaccumulating product at a high temperature and anneal primers to theproduct at a lower temperature. The transition temperatures of productdenaturation and primer annealing depend primarily on GC content andlength. If a probe is designed to hybridize internally to the PCRproduct, the melting temperature of the probe also depends on GCcontent, length, and degree of complementarity to the target.Fluorescence probes compatible with PCR can monitor hybridization duringamplification.

In accordance with the present invention, which is preferably used inconnection with rapid cycling (fully described in the above-mentionedU.S. Ser. No. 08/658,993, filed Jun. 4, 1996, entitled System And MethodFor Monitoring PCR Processes, and U.S. Ser. No. 08/537,612, filed Oct.2, 1995, entitled Method For Rapid Thermal Cycling of BiologicalSamples), a kinetic paradigm for PCR is appropriate. Instead of thinkingabout PCR as three reactions (denaturation, annealing, extension)occurring at three different temperatures for three time periods (FIG.1A), a kinetic paradigm for PCR is more useful (FIG. 1B). With a kineticparadigm, the temperature vs. time curve consists of continuoustransitions between overlapping reactions. Denaturation and annealingare so rapid that no holding time at a particular temperature isnecessary. Extension occurs over a range of temperatures at varyingrates. A complete analysis would require knowledge of all relevant rateconstants over all temperatures. If rate constants of all reactions wereknown, a “physicochemical description of PCR” could be developed.Determining these rates would require precise sample temperature controland is greatly simplified by reaction monitoring during temperaturecycling.

FIG. 2 illustrates useful temperature v. time segments for fluorescencehybridization monitoring. Product melting curves are obtained during aslow temperature increase to denaturation. By quickly lowering thetemperature after denaturation to a constant temperature, product,probe, or primer annealing can optionally be followed. Probe meltingcurves are obtained by slowly heating through temperatures around theprobe Tm. The embodiment represented in FIG. 2 provides all analysisduring temperature cycling with immediate real time display. Fluorescentprobes are included as part of the amplification solution for continuousmonitoring of primer, probe, or product hybridization during temperaturecycling.

The fluorescence hybridization techniques contained herein are based onrapid cycling, with its advantages in speed and specificity.

A sample temperature profile during rapid cycle PCR is shown in FIG. 3.

Denaturation and annealing appear as temperature “spikes” on thesefigures, as opposed to the broad plateaus of conventional temperaturecycling for PCR, e.g. FIG. 1A. Rapid temperature cycling is contrastedto conventional temperature cycling in FIG. 4, wherein it is shown that30 cycles of amplification can be completed in 15 minutes and theresulting PCR products contain many fewer side products. Thus, withrapid cycling the required times for amplification are reducedapproximately 10-fold, and specificity is improved.

EXAMPLE 1

FIG. 4 shows the results of four different temperature/time profiles(A-D) and their resultant amplification products after thirty cycles(A-D). The profiles A and B in FIG. 4 were obtained using a prior artheating block device using a prior art microfuge tube. As can be seen inFIG. 4, the transitions between temperatures are slow and manynonspecific bands are present in profiles A and B. Profile B showsimprovement in eliminating some of the nonspecific bands (in contrast toprofile A) by limiting the time each sample remains at each temperature,thus indicating that shorter times produce more desirable results.

Profiles C and D were obtained using a rapid temperature cycler. As canbe seen in FIG. 4, amplification is specific and, even though yield ismaximal with 60-second elongation times (C), it is still entirelyadequate with 10-second elongation times (D).

The optimal times and temperatures for the amplification of a 536 bpfragment of beta-globin from human genomic DNA were also determined.Amplification yields and product specificity were optimal whendenaturation (93° C.) and annealing (55° C.) were less than 1 second. Noadvantage was found to longer denaturation or annealing times. The yieldincreased with longer elongation times at 77° C., but there was littlechange with elongation times longer than 10-20 seconds. These unexpectedresults indicate that the previously available devices used for DNAamplification are not maximizing the conditions needed to optimize thephysical and enzymatic requirements of the reaction.

Further information can be obtained from: C. T. Wittwer et al., RapidCycle Allele-Specific Amplification with Cystic Fibrosis delta F(508)Locus, 39 Clinical Chemistry 804 (1993) and C. T. Wittwer et al., RapidDNA Amplification, THE POLYMERASE CHAIN REACTION 174 (1994), which areboth now incorporated herein by this reference. The instrumentation usedfor fluorescence acquisition and rapid temperature cycling is fullydisclosed in Ser. No. 08/537,612, supra.

As indicated earlier, the polymerase chain reaction can be performedrapidly. In addition to facilitating rapid heat transfer, the use ofoptically clear capillary tubes allows for continuous fluorescencemonitoring of DNA amplification in accordance with the presentinvention.

Fluorescent probes can be used to detect and monitor DNA amplification.Useful probes include double-stranded-DNA-specific dyes andsequence-specific probes. Three different fluorescence techniques forfollowing DNA amplification are compared in FIG. 5. In FIG. 5A,fluorescence depends on the hybridization of PCR product as detectedwith a double-strand-specific DNA dye. In FIG. 5B, fluorescence dependson the hydrolysis of a 5′-exonuclease quenching probe, which is wellknown in the art as discussed above. FIG. 5C diagrams a hybridizationscheme based on resonance energy transfer between fluorophores on twoadjacent probes. The method of FIG. 5A is not sequence specific,although product specificity can be determined by melting curves, oneaspect of the current invention. Both FIGS. 5B and 5C are sequencespecific. However, the hybridization method also allows analysis withmelting curves, another aspect of the current invention.

In monitoring fluorescence from reactions involving hydrolysis probes asin FIG. 5B and from reactions involving hybridization probes as in FIG.5C, it is advantageous to measure fluorescence emitted by both the donorfluorophore and the acceptor fluorophore. In practice, the majority ofthe fluorescence emitted by hydrolysis probes is from the donorfluorophore, and the majority of the fluorescence emitted byhybridization probes is from the acceptor fluorophore.

Double-strand-specific DNA dye selection. Those skilled in the art willbe familiar with the use of ethidium bromide in fluorescence techniques.When a double strand-specific fluorescent dye is present duringamplification, fluorescence generally increases as more double strandedproduct is made, see R. Higuchi et al., Simultaneous amplification anddetection of specific DNA sequences, 10 Bio/Technology 413-417 (1992). Afluorescence PCR assay for hepatitis C RNA using the intercalater,YO-PRO-1 is also known in the art. See T. Ishiguro et al., Homogeneousquantitative assay of hepatitis C virus RNA by polymerase chain reactionin the presence of a fluorescent intercalater, 229 Anal. Biochem.207-213 (1995). It is preferred that SYBR™ Green I, which is well knownin the art and available from Molecular Probes of Eugene, Oreg., be usedas a double-strand-specific dye. The molecular structure of this dye isa trade secret, but it is recommended by the manufacturer as a moresensitive double-strand-specific dye for DNA detection on gels. SYBR™Green I is heat labile, however, and thus is not useful for fluorescencemonitoring of PCR according to conventional methods where thetemperature of the reaction mixture is maintained at meltingtemperatures for extended periods of time. Because of this heatlability, it was unexpected to discover that SYBR™ Green I can be usedto monitor PCR reactions when melting temperatures are not maintainedfor extended periods, i.e. when PCR is carried out by rapid cyclingaccording to the kinetic paradigm described above.

EXAMPLE 2

Different double-strand-specific DNA dyes were compared by monitoringthe amplification of a 110 base pair fragment from the PC03/PC04 primerpair of the human beta-globin gene from 10,000 template copies. Primerswere synthesized by standard phosphoramidite chemistry as known in theart, namely, using Pharmacia Biotech Gene Assembler Plus (Piscataway,N.J.). The human beta-globin primers PC03/PC04 (110 base pairs) aredescribed in C. T. Wittwer et al., Automated polymerase chain reactionin capillary tubes with hot air, 17 Nucl. Acids. Res. 4353-4357 (1989),which is now incorporated herein by reference. DNA amplification wasperformed in 50 mM Tris, pH 8.5 (25° C.), 3 mM MgCl₂, 500 μg/ml bovineserum albumin, 0.5 μM of each primer, 0.2 mM of each deoxynucleosidetriphosphate and 0.2 U of Taq polymerase per 5 μl sample unlessotherwise stated in the following examples. Purified amplificationproduct was used as DNA template and was obtained by phenol/chloroformextraction and ethanol precipitation, see D. M. Wallace, Large- andsmall-scale phenol extractions and precipitation of nucleic acids, 152Methods in Enzymology 33-48 (1987), followed by removal of primers byrepeated washing through a Centricon 30 microconcentrator (Amicon,Danvers, Mass.). Template concentrations were determined by absorbanceat 260 nm. A(260):A(280) ratios of templates were greater than 1.7.

SYBR™ Green I (Molecular Probes, Eugene, Oreg.) was used at a 1:10,000dilution, ethidium bromide was at 5 μg/ml, and acridine orange was at 3μg/ml. These concentrations were determined to be optimal concentrationsfor maximizing the fluorescence signal observed during amplification foreach dye. Excitation was through a 450-490 nm interference filter from axenon arc source, except for ethidium bromide, where a 520-550 nmexcitation was used. For SYBR™ Green I, the emmision at 520-550 wasmonitored. Ethidium bromide fluorescence was observed through a 580-620nm bandpass. The acridine orange signal was taken as the ratio of green(520-550 nm) to red (>610 nm) fluorescence. The fluorescence of thesample before amplification was compared to the fluorescence after 35cycles (94° C. max, 60° C. for 20 sec) at 60° C. The fluorescenceincrease was 5.3-fold for SYBR™ Green I, 1.7-fold for ethidium bromide,and 1.2-fold for acridine orange. In separate experiments, thefluorescence from SYBR™ Green I was stable for greater than 30 min at70° C. It is also conveniently excited with visable light and is claimedto be less of a mutagen than ethidium bromide. Background fluorescencein all cases arose primarily from the primers.

SYBR™ Green I is a preferred double-strand-specific dye for fluorescencemonitoring of PCR, primarily because of superior sensitivity, arisingfrom greater discrimination between double stranded and single strandednucleic acid. SYBR™ Green I can be used in any amplification and isinexpensive. In addition, product specificity can be obtained byanalysis of melting curves, as will be described momentarily.

Resonance energy transfer dye selection for hybridization probes.Fluorescence resonance energy transfer can occur between 2 fluorophoresif they are in physical proximity and the emission spectrum of onefluorophore overlaps the excitation spectrum of the other. Introductorytheory on fluorescence resonance energy transfer can be found in manyrecent review articles. The rate of resonance energy transfer is:

(8.785E−5)(t⁻¹)(k²)(n⁻⁴)(q_(D))(R⁻⁶)(J_(DA)),

where:

t=excited state lifetime of the donor in the absence of the acceptor;

k²=is an orientation factor between the donor and acceptor;

n=refractive index of visible light in the intervening medium;

q_(D)=quantum efficiency of the donor in the absence of the acceptor;

R=distance between the donor and acceptor (in angstroms);

J_(DA)=the integral of (F_(D))(e_(A))(W⁴) with respect to W at alloverlapping wavelengths with:

F_(D)=peak normalized fluorescence spectrum of the donor,

e_(A)=molar absorption coefficient of the acceptor (M⁻¹cm⁻¹), and

W=wavelength (nm).

For any given donor and acceptor, a distance where 50% resonance energytransfer occurs can be calculated and is abbreviated R₀. Because therate of resonance energy transfer depends on the 6th power of thedistance between donor and acceptor, resonance energy transfer changesrapidly as R varies from R₀. At 2R₀, very little resonance energytransfer occurs, and at 0.5R₀, the efficiency of transfer is nearlycomplete, unless other forms of de-excitation predominate (i.e.,collisional quenching). R₀ values for many different donor and acceptorpairs have been compiled and vary between 22 and 72 angstroms.

In double helical DNA, 10 bases are separated by about 34 angstroms. Bylabeling the bases of DNA with donor and acceptor fluorophores,resonance energy transfer has be used as a spectroscopic ruler toobserve the helical geometry of DNA and analyze the structure of afour-way DNA junction. Resonance energy transfer can also be used as amonitor of hybridization. If a labeled oligonucleotide is hybridized toa labeled template strand, R can be brought from much greater than R₀ towell below R₀, increasing resonance energy transfer dramatically.Alternately, 2 labeled probes can be hybridized to the same templatestrand for a similar change in fluorescence energy transfer.

The practical use of resonance energy transfer to monitor hybridizationdepends on the sensitivity required and how much time is available.Using a competitive hybridization technique with 1 nM labeled probes,PCR-amplified DNA was detected after 15 min at 40° C. Faster signalgeneration is desirable. If only seconds were required forhybridization, PCR products could conveniently be quantified each cycleof amplification. Even further, the extent of probe hybridization couldbe monitored within a temperature cycle.

Hybridization is a second order process (see B. Young & M. Anderson,Quantitative analysis of solution hybridization, In: Nucleic AcidHybridization: A Practical Approach 47-71, (B. Hames, S. Higgins eds.,1985). When the concentration of the probe is much greater than theconcentration of the target, the hybridization rate is inverselyproportional to concentration of probe. For example, by doubling theprobe concentration, the hybridization time is cut in half. High probeconcentrations would be necessary for cycle-by-cycle monitoring duringPCR, because hybridization must occur before the hybridization site iscovered by polymerase extension.

The high probe concentrations required for hybridization monitoringduring PCR require a resonance energy transfer pair with uniquecharacteristics. Consider excitation of a donor (D) and an acceptor (A)pair with light. The number of fluorophores of D and A directly excitedwill be proportional to the extinction coefficient (e) of eachfluorophore at the excitation wavelength, or:

Number of D molecules directly excited=(K)(e _(D))

Number of A molecules directly excited=(K)(e _(A))

where K is a proportionality constant. De-excitation of the donor willoccur by fluorescence, resonance energy transfer, and other processessummarized as thermal quenching. If p_(F)=probability of resonanceenergy transfer, and p_(TD)=probability of donor thermal quenching, thenthe probability of donor fluorescence is:

1−p_(F)−p_(TD)

and the number of fluorescing donor molecules is:

(K)(e_(D))(1−p_(F)−p_(TD))

If the probability of detecting a donor emission in the donor emissionwindow (for example, a bandpass filter window) is PDD, then the numberof observed donor emissions is:

(p_(DD))(K)(e_(D))(1−p_(F)−p_(TD))

Now, the number of excited acceptor fluorophores is the sum of thosedirectly excited and those excited through resonance energy transfer:

(K)(e_(A))+(K)(e_(D))(p_(F))

If p_(TA)=the probability of thermal quenching of the acceptor, then theprobability of acceptor fluorescence is:

1−p_(TA)

and the number of fluorescing acceptor molecules is:

[(K)(e_(A))+(K)(e_(D))(p_(F))][1−(p_(TA))]

If the probability of detecting an acceptor emission in the acceptoremission window is p_(AA), then the number of observed acceptoremissions is:

(p_(AA))[(K)(e_(A))+(K)(e_(D))(p_(F))][1−(p_(TA))]

Finally, if the probability of observing a donor emission in theacceptor emission window is p_(DA), then the number of observedemissions (both D and A) in the acceptor emission window is:

(p_(AA))[(K)(e_(A))+(K)(e_(D))(p_(F))][1−(p_(TA))]+(p_(DA))(K)(e_(D))(1−p_(F)−p_(TD))

Since fluorescence measurements are relative and K is present in allterms, if we remove K and rearrange, the observed intensity at the donorwindow is proportional to (donor excitation)—(energy transfer):

(e_(D))(p_(DD))(1−p_(TD))−(e_(D))(p_(DD))(p_(F))  1)

and the observed intensity at the acceptor window is proportional to(acceptor excitation)+(energy transfer)+(donor emission in the acceptorwindow):

(e_(A))(p_(AA))(1−p_(TA))+(e_(D))(p_(DD))(p_(F))(1−p_(TA))+(e_(D))(p_(DA))(1−p_(TD)−p_(F))  2)

As resonance energy transfer increases, the donor signal decreases andthe acceptor signal increases. The percent signal change depends on thebackground light intensity in each window. With high concentrations ofprobes, this background light intensity is high. During PCR, whenvarying target (product) concentrations need to be monitored, it is notpossible to match the donor concentration to the target concentration.The excess donor molecules contribute to the background light intensityin both the donor and acceptor windows and partially mask the energytransfer phenomena. Background light in the acceptor window comes fromnot only donor emission in the acceptor window, but also from directexcitation of the acceptor. This background can be minimized with a lowe_(A) and a low P_(DA).

The fluorescein/rhodamine fluorescence energy transfer pair, commonlyused for nucleic acid detection, has high background fluorescence. Bothdirect acceptor excitation (e_(A,), ca. 10% e_(MAX)) and emission of thedonor at wavelengths used to detect acceptor emission (P_(DA), ca. 20%peak emission) are high. This pair can be used to monitor hybridizationif the probe concentration is near to the target concentration andenough time is allowed for complete hybridization. It is not a usefulpair of fluorophores for continuous monitoring of PCR because high probeconcentrations are required and the template concentration in PCR iscontinually changing.

Monitoring product concentration during PCR by hybridization has notbeen possible in the past because an acceptable resonance energytransfer pair had not been found. There have been few attempts to useresonance energy transfer for direct “noncompetitive” detection ofhybridization. For example, U.S. Pat. No. 5,565,322 states “the observedenergy transfer efficiency in terms of re-emission by the acceptor wasrelatively low.” At probe concentrations that are high enough forsignificant hybridization to occur in seconds, the backgroundfluorescence is too high.

Fluorescein is perhaps the most widely used fluorophore. Its extinctioncoefficient and quantum efficiency are high and it is extensively usedin microscopy, immunoassays, and flow cytometry. It is the donor in acommonly used resonance energy transfer pair with rhodamine. Cy5 is apopular red-emitting fluorophore with a very high extinctioncoefficient. The structure of the N-hydroxysuccinimide ester of Cy5 isshown in FIG. 6, and the structure of the related dye, Cy5.5, is shownin FIG. 7. These dyes are indodicarbocyanine dyes that are used commonlyin flow cytometry and automated fluorescence sequencers and areavailable from Amersham (Pittsburg, Pa.). Both fluorescein and Cy5 arecommercially available as amidites for direct, automated incorporationinto oligonucleotides. However, Cy5 has never been reported as aresonance energy transfer pair with fluorescein. Intuitively,fluorescein emission and Cy5 absorption do not overlap enough forresonance energy transfer to be considered. The emission spectrum offluorescein and absorption spectrum of Cy5 attached to oligonucleotidesare shown in FIG. 8. When the areas under the curves are normalized, theoverlap from the technical spectra is 19%. Cy5.5 excitation is shiftedto the red by about 25 nm, further decreasing the overlap withfluorescein emission to about 15%. Working in the red/infrared region ofthe spectrum is advantageous when choosing optical components forinstrumentation. Laser diodes can be used for illumination, photodiodedetectors have excellent sensitivity, and most materials have minimalautofluorescence in the pertinent spectral region.

Despite low spectral overlap, it has been discovered that fluoresceinand either Cy5 or Cy5.5 make an excellent resonance energy transfer pairfor hybridization monitoring during PCR.

EXAMPLE 3

A 110 bp beta-globin fragment was amplified from 50 ng human genomic DNAaccording to the procedure of Example 2 with the internal probesCAAACAGACACCATGGTGCACCTGACTCCTGAGGA-fluorescein (SEQ ID NO:3) andCy5-GAAGTCTGCCGTTACTGCCCTGTGGGGCAA G-p (SEQ ID NO:18) at 0.2 μM each and0.8 U KlenTaq1 polymerase (a 5′-exonuclease deficient variant of Taqpolymerase—U.S. Pat. No. 5,436,149) in a 10 μl reaction. The probeshybridized internal to the primers on the same strand and wereimmediately adjacent without any intervening bases.

Probes and primers were synthesized by standard phosphoramiditechemistry as known in the art, using a Pharmacia Biotech Gene AssemblerPlus (Piscataway, N.J.). The 3′-fluorescein-labeled probe wassynthesized on a fluorescein-labeled CPG cassette (Glen Research,Sterling, Va.) with the final trityl-ON to assist with C18 reverse phaseHPLC purification. The late eluting peak was collected and the tritylgroup was removed on a PolyPack (Glen Research). The fluorescein-labeledoligo was eluted with 50% acetonitrile and again purified by C18 reversephase HPLC. The 5′-Cy5-labeled probe was synthesized with a chemicalphosphorylation agent on the 3′-end (Glen Research) and adding a Cy5amidite (Pharmacia) to the 5′-end during trityl-OFF synthesis. Failuresequences were removed by C18 reverse phase HPLC. Probe purity waschecked with polyacrylamide electrophoresis and the absorbance of thedye and the oligo.

HPLC was performed on a 4×250 mm Hypersil ODS column (Hewlett Packard)with a 0.1 M triethanolamine:acetate mobile phase and an acetonitrilegradient at 1 ml/min. The eluate was monitored for both absorbance(A₂₆₀) and fluorescence (490 nm excitation, 520 nm emission forfluorescein and 650 nm excitation, 670 nm emission for Cy5). Tritylated-and fluorescein-labeled oligonucleotides were eluted with a 10-20%acetonitrile gradient, and Cy5-labeled oligonucleotides eluted over a10-40% acetonitrile gradient.

Temperature cycling was 94° C. for 0 sec with a programmed approach rateof 20° C./sec, 60° C. for 20 sec with an approach rate of 20° C./sec,and 75° C. for 0 sec an approach rate of 1° C./sec in a capillaryfluorescence rapid temperature cycler. During temperature cycling,fluorescein and Cy5 fluorescence were acquired each cycle at the end ofthe annealing/extension segment. Resonance energy transfer was observedas both a decrease in fluorescein fluorescence, and an increase in Cy5fluorescence beginning around cycle 26 of amplification (FIG. 9). Ingeneral, observing the fluorescence ratio of Cy5 to fluoresceinfluorescence is perferred.

The unexpectedly good results with the fluorescein/Cy5 pair can at leastpartly be rationalized. The overlap integral, J_(DA) depends not only onspectral overlap, but also on the extinction coefficient of the acceptor(Cy5 has an extinction coefficient of 250,000 M⁻¹cm⁻¹ at 650 nm), and onthe 4th power of the wavelength. Both of these factors will favor a highJ_(DA) for Cy5, even given low spectral overlap. Recently, phycoerythrinand Cy7 were shown to be an effective tandem probe forimmunofluorescence, despite low spectral overlap. In a later example,the utility of fluorescein and Cy5.5 as labels on hybridization probesis demonstrated. Fluorescence resonance energy transfer can be used tomonitor nucleic acid hybridization even when the interacting dyes havelow spectral overlap. The use of fluorescein with Cy5, Cy5.5 and otherred or infrared emitting dyes as resonance energy transfer pairs formonitoring hybridization has not been previously recognized. Fluoresceinhas a long emission “tail” that goes out to 600 nm, 700 nm and beyondthat can be used to excite these far red and infrared dyes. The rate ofenergy transfer is dependent on the overlap integral, but is alsoeffected by the 6th power of the distance between the fluorophores. Ifthe probes are designed so that the resonance energy transfer dyes arein close proximity, the transfer rate is high. At least withfluorescein/Cy5, fluorescein/Cy5.5 and like pairs, resonance energytransfer appears to predominate over collisional quenching and otherforms of energy loss when the fluorophores are close together, as in theabove example where the fluorophores are attached to adjacent probeswith no intervening bases.

The potential usefulness of a resonance energy transfer pair forhybridization probes can be judged by observing the change in the ratioof light intensity in the donor and acceptor windows at minimal andmaximal resonance energy transfer. One way to obtain minimal and maximaltransfer is to attach both fluorophores to the same oligonucleotide andmeasure fluorescence ratio before and after digestion withphospodiesterase.

EXAMPLE 4

The dual-labeled fluorescein/Cy5 probeCy5-CTGCCG-F-TACTGCCCTGTGGGGCAAGGp (SEQ ID NO:19) was synthesized bystandard phosphoramidite chemistry, where p is a terminal 3′-phosphate(chemical phosphorylation reagent, Glen Research), F is a fluoresceinresidue introduced as an amidite with a 2-aminobutyl-1,3-propanediolbackbone to maintain the natural 3-carbon internucleotide phosphodiesterdistance (ClonTech, Palo Alto, Calif.), and Cy5 is added as the amidite(Pharmacia). The ratio of Cy5 to fluorescein fluorescence in 0.1 M Tris,pH 8.0 was obtained before and after exhastive hydrolysis withphosphodiesterase (Sigma, St. Louis, Mo.). The change in thefluorescence ratio was 220-fold after hydrolysis. A dual-labeledfluorescein/rhodamine probe F-ATGCCCT*CCCCCATGCCATCCTGCGTp (SEQ IDNO:20) was purchased from Perkin Elmer (Foster City, Calif.), where F isfluorescein and * is a rhodamine attached to a modified T residue by anamino-linker arm. The change in the fluorescence ratio (rhodamine tofluorescein) was 22-fold after hydrolysis with phosphodiesterase.

The potential signal from the fluorescein/Cy5 pair was 10-fold that ofthe fluorescein/rhodamine pair.

EXAMPLE 5

The effect of the ratio, concentration, and spacing of fluorescein andCy5-labeled adjacent hybridization probes during PCR was studied.Amplification of the beta globin locus and probe pair of Example 3 wasused and the maximum change in the fluorescence ratio of Cy5 tofluorescein was observed. The maximal signal occurred when the ratio ofCy5 to fluorescein-labeled probes was 2:1 (FIG. 10). At this 2:1 ratio,the best signal occurred at a fluorescein probe concentration of 0.2 μMand a Cy5-labeled probe concentration of 0.4 μM (FIG. 11). The optimalnumber of intervening bases between adjacent hybridization probes duringPCR was also determined. Several probes of the same length but slightlyshifted in their hybridization position were synthesized according toExample 3 so that when they hybridized to the beta globin target, 0, 1,2, 3, 4, or 6 bases remained between the probes. The highest signalduring PCR occurred with one intervening base (FIG. 12). Although someresonance energy transfer was detected at a spacing of 15 and even 25bases, much better transfer occurred at 0-5 bases.

Heller et al. (U.S. Pat. No. 4,996,143), found that energy transferefficiency decreased as the number of nucleotides between fluorophoresdecreased from 4 to 0 units. In contrast, the best energy transfer withthe fluorescein/Cy5 pair was seen at 0 to 2 intervening nucleotides.

Hybridization probe method. If 2 probes are synthesized that hybridizeadjacently on a target and each is labeled with one fluorophore of aresonance energy transfer pair, the resonance energy transfer increaseswhen hybridization occurs (FIG. 5C). The fluorescein/rhodamine pair ismost commonly used for nucleic acid detection.

One aspect of this invention is to provide a sequence-specifichomogeneous hybridization method for detection of PCR products. It isnot obvious how to achieve this. Using hybridization probes duringamplification is counterintuitive. It does not seem that both probehybridization and polymerase extension can occur. To get sequencespecific fluorescence, the probes must be hybridized, but the probescannot be hybridized if the polymerase is to complete primer extensionand exponentially amplify DNA.

One solution to this problem is to use a dual-labeled single probe andutilize the 5′-exonuclease activity of common heat stable DNApolymerases to cleave the probe during extension, thereby separating the2 fluorophores. In this case, the fluorescence signal arises fromseparation of the resonance energy transfer pair upon probe hydrolysis(FIG. 5B), rather than approximation of the fluorophores by adjacenthybridization (FIG. 5C). However, dual-labeled probes are difficult tomake, requiring manual addition of at least one fluorophore to the oligoand usually require extensive purification. The probes are expensive,and two dual-labeled probes are necessary for competitive quantificationof a target or for mutation detection. A further concern is that theobserved fluorescence depends on the cumulative amount of probehydrolyzed, not directly on the amount of product present at any givencycle. This results in a continued increase in fluorescence even afterthe PCR plateau has been reached. Finally and most importantly, probehydrolysis does not always occur during polymerase extension, an effectthat is not well understood. For example, the dual-labeledfluorescein/Cy5 probe of Example 4 showed very poor hydrolysis duringPCR when it was flanked by primers. Indeed, several dual-labeledfluorescein/Cy5 probes, including those with terminal labels, were madeand all showed poor hydrolysis and signal generation duringamplification.

Homogeneous detection of PCR products with adjacent hybridization probeswould solve many of the problems of the 5′-exonuclease system. Synthesisof adjacent hybridization probes is relatively simple because amiditesfor both fluorescein and Cy5 are available for direct incorporationduring automated synthesis and dual labeling of one probe is notrequired. Because their fluorescence results from hybridization, nothydrolysis, the temperature dependence of probe fluorescence could beused for mutation detection and quantification. However, use of adjacenthybridization probes for homogeneous detection of PCR products has notbeen reported previously. Surprisingly, both hybridization for signalgeneration and amplification by polymerase extension through the areablocked by the probes can occur.

EXAMPLE 6

A 110 bp beta-globin fragment was amplified from genomic DNA withadjacent fluorescein- and Cy5-labeled probes as described in Example 3.Either 0.4 U (Taq) or 0.8 U (Stoffel fragment, Perkin Elmer, orKlenTaq1) of enzyme was used in 10 μl reactions. Unless indicatedotherwise, temperature cycling was 94° C. for 0 sec with a programmedapproach rate of 20° C./sec, 60° C. for 20 sec with an approach rate of20° C./sec, and 75° C. for 0 sec with an approach rate of 1° C./sec.FIG. 13 shows the development of fluorescence by 2 adjacenthybridization probes immediately after the template was amplified for 30cycles. After a brief denaturation at 94° C., the temperature waslowered to 60° C. and fluorescence increased for about 20 sec. Themagnitude of the signal is greater with an exonuclease deficientpolymerase (Stoffel fragment) than with native Taq polymerase thatincludes a 5′-exonuclease activity. After about 20 sec., thefluorescence drops as the polymerase displaces and/or hydrolyzes theprobes. The relative decrease in fluorescence is slightly faster whenthe polymerase has 5′-exonuclease activity (Taq DNA polymerase) thenwhen it lacks this activity (Stoffel fragment).

In FIG. 14 (top panel), the temperature is cycled between 94° C. and 60°C. with a 20 sec hold at 60° C. Fluorescence is acquired at the end ofthe 20 sec when fluorescence is maximal. Good amplification occurs withTaq (exo⁺ ), but not with Stoffel fragment (exo⁻) as verified by bothfluorescence development and agarose gels (gels not shown). However, ifthe time at 60° C. is increased from 20 sec to 120 sec (FIG. 14, middlepanel), the exo⁻ polymerase amplifies well. The slower rate of probedisplacement with an exo⁻ polymerase apparently requires more time at60° C. for efficient amplification than the exo⁺ polymerase. The timerequired by exo⁻ polymerases can be reduced by slowly increasing thetemperature from 60° C. to 75° C. (FIG. 14, bottom panel). Thepolymerase stalls when it reaches the probe. However, at the probemelting temperatures, the probes melt off the template and thepolymerase continues unencumbered to complete polymerization of thestrand. Polymerization is completed as long as the temperature is notraised too quickly after probe melting. FIG. 14 (bottom panel) shows oneexo⁺ polymerase (Taq) and two exo⁻ polymerases (Stoffel fragment andKlenTaq1).

When exonuclease activity is present, some of the probe is hydrolyzedeach cycle as evidenced by an the decrease in fluorescence withextensive amplification. This is observed in FIGS. 13 and 14 (middle andbottom panels), but is does not occur with exo⁻ polymerases. Because thefluorescence is stable on extensive amplification, exo⁻ polymerases suchas KlenTaq1 are preferred.

The success of using adjacent hybridization probes to monitor PCRdepends on several factors. Resonance energy transfer is maximized whenthere is either 0 to 2 intervening bases between adjacent hybridizationprobes. To increase the fraction of strands that hybridize to the probesbefore the primer extends through the area of probe hybridization, theprobe melting temperatures should be greater than the primer meltingtemperatures (preferably >5° C.).

Cycle-by-cycle fluorescence. Conventional endpoint analysis of DNAamplification by gel electrophoresis identifies product size andestimates purity. However, because amplification is at first stochastic,then exponential, and finally stagnant, the utility of endpoint analysisis limited for quantification. One aspect of the present inventionincludes cycle-by-cycle monitoring for quantification of initialtemplate copy number with hybridization probes. As will be appreciatedby those skilled in the art, once-per-cycle monitoring of multiplesamples undergoing DNA amplification is a powerful quantitative tool.Cycle-by-cycle monitoring is achieved by acquiring fluorescence duringthe extension or combined annealing/extension phase of each cycle andrelating the fluorescence to product concentration.

EXAMPLE 7

Cycle-by-cycle monitoring of PCR was performed by three differentfluorescence techniques. Fluorescence was monitored by (i) thedouble-strand-specific dye SYBR™ Green I, (ii) a decrease in fluoresceinquenching by rhodamine after exonuclease cleavage of a dual-labeledhydrolysis probe and (iii) resonance energy transfer of fluorescein toCy5 by adjacent hybridization probes. Amplification reagents andconditions were as described in Example 2. The human beta-globin primersRS42/KM29 (536 base pairs) and PC03/PC04 (110 base pairs) are describedin C. T. Wittwer et al., Automated polymerase chain reaction incapillary tubes with hot air, 17 Nucl. Acids. Res. 4353-4357 (1989),which is now incorporated herein by reference. Temperature cycling forbeta-globin was 95° C. maximum, 61° C. minimum, 15 sec at 72° C. and anaverage rate between temperatures of 5.2° C./sec. The beta-actin primersand fluorescein/rhodamine dual probe were obtained from Perkin Elmer(no. N808-0230). Temperature cycling for beta-actin was 94° C. maximum,60° C. for 15 sec with an average rate between temperatures of 6.2°C./sec. The single labeled probes5′-CAAACAGACACCATGGTGCACCTGACTCCTGAGGA-fluorescein-3′ (SEQ ID NO:3) and5′-Cy5-AAGTCTGCCGTTACTGCCCTGTGGGGCAAGp (SEQ ID NO:4) were synthesized asin Example 3. These adjacent probes hybridize internal to the PC03/PC04beta-globin primer pair on the same DNA strand and are separated by onebase pair. Temperature cycling was 94° C. maximum, 59° C. for 20 secwith an average rate between temperatures of 7.0° C./sec. Hybridizationprobes (beta-actin and beta-globin) were used at 0.2 μM each.

When multiple samples are monitored once each cycle with SYBR™ Green I,a 10⁷-10⁸ range of initial template concentration can be discerned asrepresented in FIG. 15. This amplification is of a 536 base pairfragment of the beta-globin gene, with SYBR™ Green I as thedouble-strand specific dye. When the data were normalized as the percentmaximal fluorescence of each sample, one hundred initial copies wereclearly separated from ten copies. However, the difference between oneand ten copies was marginal, and no difference was observed between zeroand one average copies per sample.

In contrast, sequence-specific probes have a similar dynamic range but,appear to discriminate even a single initial template copy from negativecontrols. Signal generation with 5′-exonuclease probes (beta-actinfragment, FIG. 16) is dependent not only on DNA synthesis, but requireshybridization and hydrolysis between the fluorophores of thedual-labeled probe. This hydrolysis reduces quenching and thefluorescence ratio of fluorescein to rhodamine emission increases.Whereas the fluorescence from double strand dyes levels off with excesscycling (FIG. 15), the signal from exonuclease probes continues toincrease with each cycle (FIG. 16). Even though no net product is beingsynthesized, probe hybridization and hydrolysis continue to occur. Asthe template copy number decreases below 10³, signal intensitydecreases, but low copy numbers can still be quantified because thenegative control signal is stable.

In FIG. 17, amplification is monitored using adjacent hybridizationprobes and is expressed as a ratio of Cy5 to fluorescein fluorescence.The change in fluorescence ratio is largely due to an increase in Cy5fluorescence from resonance energy transfer (FIG. 9). In contrast todual-labeled hydrolysis probes, the fluorescence signal of hybridizationprobes decreases at high cycle numbers if the polymerase contains anexonuclease activity (see also FIG. 14).

The present invention's feasibility using two different methods forresonance energy transfer detection of hybridization during PCR will nowbe demonstrated. The first method uses two adjacent hybridizationprobes, one labeled 3′ with fluorescein and the other labeled 5′ withCy5. As product accumulates during PCR, the probes hybridize next toeach other during the annealing segment of each cycle. The second methoduses a primer labeled with Cy5 and a single hybridization probe. Thelabeled primer is incorporated into the PCR product during amplificationand only a single hybridization is necessary.

EXAMPLE 8

Cycle-by-cycle monitoring of PCR was performed by resonance energytransfer between a Cy5-labeled primer and a fluorescein-labeledhybridization probe. This was compared to monitoring with adjacentCy5/fluorescein hybridization probes. The Cy5-labeled primer wasCAACTTCATCCACGT*TCACC (SEQ ID NO:21) where T* is a modified T base withCy5 attached and the corresponding probe wasGTCTGCCGTTACTGCCCTGTGGGGCAA-fluorescein (SEQ ID NO:22). The adjacenthybridization probes were CCTCAAACAGACACCATGGTGCACCTGACTCC-fluorescein(SEQ ID NO:23) and Cy5-GAAGTCTGCCGTTACTGCCCTGTGGGGCAAp (SEQ ID NO:24).The hybridization probes were synthesized according to Example 3 andused at 0.2 μM. The Cy5-labeled primer was synthesized in two steps.Automated synthesis was used to incorporate an amino-modifier C6dT (GlenResearch) at the desired T position. Then, the monovalentN-hydroxysuccinimide ester of Cy5 (FIG. 6) was manually conjugated tothe amino linker according to the manufacturer's instructions(Amersham). HPLC purification was as described in Example 3.

The Cy5-labeled primer (0.5 μM) was used instead of PC04 to amplify the110 base pair beta-globin fragment from human genomic DNA as in Example3, except that 0.4 U of Taq polymerase was used per 10 μl. The adjacenthybridization probes also monitored amplification of the samebeta-globin fragment. Temperature cycling was done at 94° C. for 0 secand 60° C. for 20 sec. The fluorescence was monitored once each cycle atthe end of the annealing/extension segment. In both methods,fluorescence energy transfer to Cy5 increases with hybridization and isplotted as a ratio of Cy5 to fluorescein fluorescence (FIG. 18).

In additional experiments, the number of bases separating the Cy5-labeland the fluorescein label were varied. The best fluorescence resonanceenergy transfer was observed with about 4-6 bases between thefluorophores, although a signal was detectable up to at least 15 bases.

In contrast to hydrolysis probes, the fluorescence signal fromhybridization probes is not cumulative and develops anew during eachannealing phase. The fluorescence is a direct measure of productconcentration because the hybridization is a pseudo-first orderreaction. Because the concentration of probe is much greater than theproduct, the fraction of product hybridized to probe is independent ofproduct concentration. These characteristics indicate that using asingle hybridization probe along with a labeled primer will provide asuperior monitor of product accumulation for quantification. Theinherent variance of different fluorescence techniques duringcycle-by-cycle monitoring is also important for quantification.

EXAMPLE 9

DNA amplification was performed according to Example 2 for each of threedifferent fluorescence monitoring methods. SYBR™ Green I was used at a1:10,000 dilution in the amplification of a 205 base pair humanbeta-globin fragment from primers KM29 and PC04. The hydrolysis probeand conditions are those specified in Example 7. The hybridizationprobe, TCTGCCGTTACTGCCCTGTGGGGCAAG-fluorescein (SEQ ID NO:5) was usedwith KM29 and the Cy5-labeled primer CAACTTCATCCACGTT*CACC (SEQ ID NO:6)where T* was a Cy5-labeled T base synthesized as in example 8. Allamplifications were performed in ten replicates with 15,000 templatecopies (50 ng of human genomic DNA/10 μl). The temperature cycles were31 sec long (94° C. maximum, 60° C. for 20 sec, average rate betweentemperatures 6.2° C./sec). Fluorescence was acquired for each samplebetween seconds 15 and 20 of the annealing/extension phase.

FIG. 19 allows comparison of three fluorescence monitoring techniquesfor PCR. The fluorescence probes are the dsDNA dye SYBR™ Green I (FIG.19A), a dual-labeled fluorescein/rhodamine hydrolysis probe (FIG. 19B),and a fluorescein-labeled hybridization probe with a Cy5-labeled primer(FIG. 19C). All probes had nearly the same sensitivity with detectablefluorescence occurring around cycle 20. With extended amplification, thesignal continued to increase with the hydrolysis probe, was level withSYBR™ Green I, and slightly decreased with the hybridization probe. Theprecision of the three fluorescence monitoring techniques are comparedin FIG. 19D. The mean +/− standard deviations are plotted for eachpoint. The data are plotted as the coefficient of variation (standarddeviation/mean) of the fluorescence ratio above baseline (taken as theaverage of cycles 11-15).

Although the change in fluorescence ratio from the hydrolysis probe isgreater than that from the hybridization probe (FIGS. 19B and 19C), thecoefficient of variation of fluorescence from the hydrolysis probes isgreater (FIG. 19D). That is, the fluorescence resulting from thehybridization probe method is more precise than using a hydrolysisprobe, even though the absolute signal levels are lower. This is anunexpected advantage of hybridization probes over the more usualdual-labeled hydrolysis probes.

Quantification of initial template copy number. Quantitative PCR hasbecome an important technique in both biomedical research and in theclinical laboratory. The process of quantification often includesrunning a standard curve of samples containing known copy numbers of thetarget sequence. The copy number of an unknown sample is determined byextrapolation between the known values. When a complete amplificationcurve is monitored cycle-by-cycle using fluorescence, radioactivity orany other method that gives a signal proportional to the amount of DNApresent, many data points are available for analysis and it is notobvious which value to choose to represent a standard or unknown. Priorart is to choose a “threshold value” of the signal and then use thecycle number when the standard or unknown crosses that threshold as therepresentative value (see Higuchi & Watson, EPA 0 640 828 A1). Thisapproach uses a very small amount of the available data in anamplification curve. In addition, the assignment of the threshold valueis highly subjective and is subject to conscious or unconscious bias.More of the available data could be used objectively by applyingnon-linear curve fitting techniques to the data in an amplificationcurve. Preferably, equations could be found that describe the shape ofthe amplification curves by modeling factors of the underlying process.

A number of different equations could be used to fit the data producedduring amplification. DNA amplifications typically have a log linearsegment and the data in this segment can be fit to an equation thatdescribes an exponential increase like that expected in a DNAamplification. The log-linear portion of a DNA amplification can bedescribed by the equation:

y=A*[DNA]*(1+E)^(n)

wherein A is a scaling factor that converts units of signal to units ofDNA; [DNA] is the starting concentration of DNA in the reaction;

E is the efficiency of the reaction; and n is the cycle number.

A quantification process would involve: (1) fitting the known standardsto this equation allowing the parameters A and E to float, and (2)fitting the unknown samples to the equation using the values of A and Efrom the standards and allowing [DNA] to float. This technique uses muchmore of the data and uses the portion of the data, the log-linearportion, that is likely to be most informative. FIGS. 20, 21 and 22 showan example of this approach. Ten-fold dilutions of a purified PCRproduct were amplified as a standard curve and an “unknown” humangenomic DNA standard was used. FIG. 20 shows that the log-linear portionis easily identified either by the user or by software. FIG. 21 shows afit of the equation y=A*[DNA]*(1+E)^(n) to the 10⁴ copy standard. FIG.22 uses average values from several standards for A and E and fits[DNA]. The fit value of 16,700 is very close to the theoretical valuefor a single copy gene in genomic DNA (15,000 copies).

Using all the data in an amplification curve would include thebackground level and the plateau value. While at high copy number theplateau is uninformative, at low copy number it is often proportional tostarting copy number. The background level could be useful indetermining the first point that shows a significant increase in signal.At this time all the factors involved in the shape of the DNAamplification curve are not known, so one approach is to describe theshape of the curve. FIG. 23 shows amplification curves using fluorescenthybridization probes to detect a five order of magnitude range of DNAtemplate concentrations. Each curve is fit to the equation:

y=((as*x+ab)−(ds*x+db))/(1+(x/c){circumflex over ( )}b)+(ds*x+db)

wherein “as” is the background of the slope line, “ab” is the yintercept of the background line, “ds” is the slope of the plateau line,“db” is the y intercept of the slope line, “c” is cycle number where thereaction is half way from background to plateau (A₅₀), and “b” is theslope of the log-linear portion of the amplification.

This equation gives good fits to this amplification data, and FIG. 24shows that the value of the A₅₀ correlates well with the log of thestarting copy number across seven orders of magnitude. FIG. 25 shows thesame equation fit to data from amplifications that used a hydrolysisprobe to detect DNA template over a 5 order of magnitude range. Thisequation gives good fits to this amplification data, and FIG. 26 showsthat the value of the A₅₀ correlates well with the log of the startingcopy number. This demonstrates the flexibility of the full curve fitapproach as the equation has given good fits to both the sharp plateausof the hybridization probe amplification curves and the steadilyincreasing “plateaus” of the hydrolysis probe curves.

Total curve fits are not limited to this equation. FIG. 27 shows anamplification of three concentrations of DNA template fit to theequation: y=(((as*x+ab)−(dmax*x/dd+x))/(1+(x/c){circumflex over ()}b))+(dmax*x/dd+x), which is similar to the first 6 parameter equationsexcept that the plateau is defined by a hyperbolic curve rather than bya line. FIG. 28 shows that the A₅₀ for this equation correlates well tothe starting copy number.

While the A₅₀ has been used in these examples and level between thebackground and the plateau could be chosen if a particular technique ismore robust lower or higher in the amplification profile. For example aseries of amplification standard curves are evaluated for the bestcorrelation between the starting copy number and the A₅₀, the A₄₀, theA₃₀, the A₂₀, and the A₁₀. The level of amplification that bestcorrelates with the known starting copy number is determined. This willbe different for different detections systems. FIG. 19 shows thatcoefficient of variation for various detection systems. The level ofamplification that is the best predictor is likely to be the level withthe lowest coefficient of variation.

As the DNA amplification reaction itself is better understood, otherequations that have parameters that reflect physical processes could beused. The plateau of the DNA amplification curve has different causes indifferent reactions. It is often due to the inability of the primers tocompete with product reannealing in the latter cycles. This effect couldbe captured with a parameter that is dependent on the square of theconcentration of product in the reaction (as reannealing rate isproportional to the square of the product concentration). Another causeof the plateau can be the depletion of the primers. Primer limitedreactions have a characteristic shape, they have a very sharp plateauthat can be recognized. Primer limited reaction fits will includeparameters that define this sharp top. Enzyme limited reactions have avery rounded plateau that can be fit accordingly. Weighting factors canbe devised that reflect the known coefficients of variation for thegiven system to more heavily weight the more reliable data points. Byfitting more of the points in an amplification profile, more accurateand robust estimates of starting copy number can be obtained. One ormore of the parameters of these fits can be used to estimate thestarting copy number of unknown samples.

Continuous fluorescence monitoring of PCR. The present invention'sfeature of continuous monitoring, that is, monitoring many times withineach PCR cycle, will now be discussed. While fluorescence monitoringduring PCR can be done once each cycle at a constant temperature, thepresent invention provides the important advantage of providingcontinuous monitoring throughout the PCR cycle. Temperature is importantbecause fluorescence changes as the temperature changes. FIGS. 29A&Bdemonstrate the inverse relationship between temperature andfluorescence for SYB™ Green I. This is a confounding effect duringtemperature cycling that is usually eliminated by consideringfluorescence once per cycle at a constant extension temperature.However, in accordance with the present invention, monitoringfluorescence during temperature changes is very informative. Prior tothe present invention, continuous fluorescence monitoring within eachcycle, as opposed to once each cycle, has not been carried out. Inaccordance with the present invention, time, temperature andfluorescence is acquired every sec, every 200 msec, every 100 msec oreven at a greater frequency. Such data can reveal fine details ofproduct denaturation, reannealing and extension, and probe annealing andmelting during rapid cycling not available in previously availablemethods.

EXAMPLE 10

A 180-base-pair fragment of the hepatitis B surface antigen gene wasamplified from 106 copies of purified PCR product using primers5′-CGTGGTGGACTTCTCTCAAT-3′ (SEQ ID NO:1), and 5′-AGAAGATGAGGCATAGCAGC-3′(SEQ ID NO:2) (Genbank sequence HVHEPB). The amplification conditions ofExample 2 were followed except that the reaction contained a 1:20,000dilution of SYBR™ Green I and 2 mM MgCl₂. Each temperature cycle was 27sec long (92° C. maximum, 59° C. minimum, 5 sec at 70° C., average ratebetween temperatures 3.0° C./sec). Time, temperature, and 2 channels offluorescence were acquired every 200 msec and continuously displayed asfluorescence v. cycle number and fluorescence v. temperature plots. FIG.30 shows a 3D trace of temperature, time and fluorescence for cycles 10through 34. This 3D curve is also projected in FIG. 30 as 2D plots oftemperature v. time, fluorescence v. time, and fluorescence v.temperature. The temperature v. time projection of FIG. 30 repeats eachcycle and provides essentially the same information as set forth in FIG.3. Because fluorescence varies inversely with temperature, thefluorescence v. time projection shown in FIG. 30 at early cycles is ascaled mirror image of the temperature v. time plot (see FIG. 29). Asproduct accumulates, the fluorescence increases at all temperatures withdouble stranded product. However at denaturation temperatures,fluorescence returns to baseline since only single stranded DNA ispresent. The fluorescence v. temperature projection of double strandeddyes shown in FIG. 30 eliminates the time axis and shows the temperaturedependence of strand status during DNA amplification.

EXAMPLE 11

A 536 base pair fragment of the human beta-globin gene was amplifiedfrom 25 ng of genomic DNA and a 1:10,000 dilution of SYBR™ Green I in avolume of 5 μl. Each temperature cycle was 28 sec long (95° C. maximum,61° C. minimum, 15 sec at 72° C. with an average rate betweentemperatures of 5.2° C./sec). Other conditions are the same as thosedescribed in FIG. 30. Cycles 15-40 are displayed. The temperaturedependence of product strand status during PCR is revealed byfluorescence v. temperature plots using as shown in FIG. 31. Earlycycles represented appear identical, with a nonlinear increase influorescence at lower temperatures. As amplification proceeds,temperature cycles appear as rising loops between annealing anddenaturation temperatures. As the sample is heated, fluorescence is highuntil denaturation occurs. As the sample cools, fluorescence increases,reflecting product reannealing. When the temperature is constant duringextension, increasing fluorescence correlates with additional DNAsynthesis.

As will be appreciated by an understanding of this disclosure,continuous monitoring within a cycle can provide insight into DNAamplification mechanics not previously available in the art. Using thepresent invention, many aspects of DNA amplification that haveheretofore been little understood are discernable. For example, rapidcycle amplification claims to denature the product in less than onesecond, while the prior art uses ten seconds to one minute ofdenaturation. Observing product melting by real time fluorescencemonitoring with double strand dyes in accordance with the presentinvention (FIGS. 30 and 31) shows that use of the shorter denaturationtimes is effective. As another example, many causes of the known“plateau effect” have been proposed, but few data are available todistinguish between alternatives. As shown in FIG. 31, productreannealing is very rapid. In fact, during later cycles ofamplification, a majority of product is reannealed each cycle duringcooling before the primer annealing temperature is reached. This occurswith cooling rates of 5-10° C./sec in rapid cycle instrumentation. Theproduct reannealing with slower, prior art temperature cyclers will bemore extensive and this undesirable effect will be greater. Productreannealing appears to be a major, and perhaps the sole, cause of the“plateau effect.”

Now consider continuous monitoring of sequence specific probes. As willbe appreciated by an understanding of this disclosure, continuousmonitoring within a cycle can identify the nature of probe fluorescence.

EXAMPLE 12

Continuous monitoring of amplification every 200 msec was performed witha dual-labeled hydrolysis probe (beta-actin) and adjacent hybridizationprobes (beta-globin) as in Example 7. In FIG. 32A, cycles 20-45 of areaction monitored with the hydrolysis probe is shown. Hydrolysis probesshow a linear change in fluorescence ratio with temperature and aparallel increase in fluorescence as more probe is hydrolyzed. Incontrast, the fluorescence ratio from hybridization probes variesradically with temperature (FIG. 32B, cycles 20-40). During theannealing/extension phase, the probes hybridize to single strandedproduct and the fluorescence ratio (Cy5/fluorescein) increases. Duringheating to product denaturation temperatures, the probes dissociatearound 70° C., returning the fluorescence ratio to background levels.

EXAMPLE 13

A 110 base pair beta-globin fragment was amplified from 50 ng of genomicDNA in a volume of 10 μl. The amplification conditions and adjacenthybridization probes of Example 3 were followed with either 0.4 U of Taqpolymerase or 0.8 U of KlenTaq1. Fluorescence was monitored each 100msec. Fluorescence v. temperature plots using KlenTaq1 (FIG. 33) and Taq(FIG. 34) demonstrate melting of the probes at about 70° C. The maximalsignal with KlenTaq1 is greater than that with Taq, because of theexonuclease activity of the latter. At later cycles with Taq, thefluorescence each cycle begins to decrease as the concentration ofintact probe decreases. Three dimensional plot of temperature, time, andfluorescence are shown in FIG. 35 (KlenTaq1) and FIG. 36 (Taq).

The present invention's combination of (1) continuous fluorescencemonitoring within each temperature cycle and (2) analysis of thetemperature and time dependence of hybridization provides advantages nototherwise obtainable. FIG. 2 shows that information that was previouslyunobtainable can be extracted by continuous monitoring throughout thecycle. Continuous fluorescence monitoring during the product meltingphase of the cycle provides useful information on the purity, identity,and quantity of DNA present during that cycle.

As a PCR reaction is heated from the extension temperature to thedenaturation temperature, any DNA in the sample is melted to singlestrands. This denaturation can be observed as a drop in the fluorescenceof SYBR™ Green I. For small PCR products, the melting transition occursover a narrow temperature range and the midpoint of that melting rangeis referred to as the Tm. Similar to sizing by gel electrophoresis,melting peak analysis measures a fundamental characteristic of DNA andcan be used to identify amplified products. Unlike gel electrophoresis,melting curve analysis can distinguish products of the same length butdifferent GC/AT ratio. In addition, two products with the same lengthand GC content, but differing in their GC distribution (for example,equally distributed vs. a GC clamp on one end) would have very differentmelting curves. The temperature at which PCR products melt varies over alarge range. Using empirical formulas known in the art, the effect of GCcontent on the melting temperature (Tm) of DNA predicts that a 0% GCduplex would melt 41° C. lower than a 100% GC duplex. Given the same GCcontent, a 40 base pair primer dimer would melt 12° C. below a 1000 bpproduct. Hence, the range of Tm for potential PCR products spans atleast 50° C. This wide range allows most PCR products to bedifferentiated by melting curves. Thus, the combination of fluorescencemonitoring of PCR with melting curve analysis provides simultaneousamplification, detection, and differentiation of PCR products.

EXAMPLE 14

DNA melting curves for three different PCR products were acquired on amicrovolume fluorimeter integrated with a 24-sample thermal cycler withoptics for SYBR™ Green I fluorescence (LightCycler LC24, IdahoTechnology, Idaho Falls, Id.). The primers for the 180 base pairhepatitis B surface antigen gene amplification were5′-CGTGGTGGACTTCTCTCAAT-3′ (SEQ ID NO:1) and5′-AGAAGATGAGGCATAGCAGC-3′(SEQ ID NO:2). The primers for the 292 basepair prostate specific antigen (PSA) gene amplification were5′-CTGTCCGTGACGTGGATT-3′ (SEQ ID NO:7) and 5′-AAGTCCTCCGAGTATAGC-3′ (SEQID NO:8). The 536 base pair human beta-globin gene amplification wasdone as in Example 7. PCR was performed as described in Example 2.Amplification products were purified by phenol/chloroform extraction andethanol precipitation, followed by repeated washing through a Centricon30 microconcentrator (available from Amicon of Danvers, Mass.). Templateconcentrations were determined by absorbency at 260 nm and hadA(260)/A(280) ratios greater than 1.7.

Fifty ng of purified DNA in 50 mM Tris, pH 8.5, 2 mM MgCl₂, and 250μg/ml bovine serum albumin and a 5 μl volume were pipetted into the openplastic reservoir of composite glass/plastic reaction tubes, centrifugedat low speed to place the sample at the tip of the glass capillary, andsealed inside with a plastic plug. Fluorescence data for melting curveswas acquired by integrating the signal over 0.25-2.0 seconds during alinear temperature transition to 95° C. at 0.1-10.0° C./second. Thefluorescence was continuously acquired and displayed at fluorescence v.temperature plots in the LabView programming environment (NationalInstrument, Austin, Tex.). FIG. 37 shows the melting curves of the threepurified PCR products.

The Tm's of three products in FIG. 37 span only 6 degrees and two of thecurves are separated by only 2 degrees. This small separation is ampleto allow easy differentiation of the products. The importance of GCpercentage over length on Tm is illustrated by the 292 bp PSA productmelting at a higher temperature than the longer 536 bp beta-globinfragment. Melting curves are often obtained at rates of 0.5° C./minuteto ensure equilibrium. Moreover, as the heating rate decreases, themelting curve shifts to lower temperatures and becomes sharper (FIG. 38,hepatitis B fragment). Note however, that the melting curves of FIG. 37were obtained during a heating rate of 0.2° C./sec (12° C./minute) andcan differentiate products differing in Tm by 2° C. or less.

The apparent Tm of PCR products is also dependent ondouble-strand-specific DNA dye concentration (FIG. 39, hepatitis Bfragment). Higher concentrations of dye increase the stability of theDNA duplex and the observed Tm.

For monitoring of melting curves with SYBR™ Green I, the preferredconditions are 1:7,000-1:30,000 fold dilution of SYBR Green I withheating rates of 0.1-0.5° C./second. These conditions allow easydifferentiation of products that differ in Tm by 2° C.

More precise temperature control and software for melting peak analysiswill reduce the detectable difference in Tm to a fraction of a degree.This will allow the differentiation of most PCR products. Not allproducts can be differentiated by Tm however, just as it is possible tomisread electrophoresis results because of comigration of two or moreproducts, it is possible that some of the product melting in theexpected range may not be the intended product. However, if no DNA meltsin the range of the expected product, it can conclusively be said thatnone of the expected product is present.

Another form of product differentiation available with melting curveanalysis is the distinctive patterns of domain melting seen in longerPCR products. While short products (<300 bp) usually melt in onetransition, longer products can have internal melting domains that givemelting curves of a complex, distinctive shape. These complex meltingcurves can be used as a fingerprint for product identification.

Melting curve analysis can be used to differentiate intended productfrom nonspecific products such as primer dimers. Primer dimers melt overa wide range of low temperatures; very different from the sharp meltingcurves of specific PCR amplification products. Larger heterogeneousproducts which resulted from running many cycles at low annealingstringency have lower and broader melting curves when compared with purePCR product.

EXAMPLE 15

Amplification of the 536 beta-globin gene fragment was performed as inExample 7 with a 1:30,000 dilution of SYBR™ Green I except that theconditions were varied. In reaction A (FIG. 40), no template was addedand the reaction was cycled at 94° C. for 0 sec, 60° C. for 0 sec, and72° C. for 10 sec for 30 cycles to p small nonspecific amplificationproducts. In B, amplification of 10⁶ initial copies of purified templateat low stringency (94° C. for 0 sec, 50° C. for 0 sec, and 72° C. for 10sec) for 55 cycles showed a broad size range of amplification productson gel electrophoresis and melts across a wide temperature range. In C,10⁶ initial copies of purified template were cycled at 94° C. for 0 sec,60° C. for 0 sec, and 72° C. for 10 sec for 30 times and shows a singlebright band and melts in a sharp transition. The temperature transitionrate was 0.2° C./sec. A Hind III digest of λ phage DNA (M) is used as amarker.

FIG. 40 shows how melting curves accurately reflect the specificity of aPCR reaction. The sharp, high temperature melting curve C corresponds toa single band on a gel. The low temperature, broad melting, curve Acomes from analysis of a no template control that shows only primerdimers. Over-amplification of the product in C gives the intermediatemelting curve B, still clearly differentiable from the specific product.

The melting curves seen, for example, in FIG. 37, can be betterquantified by first taking the derivative of fluorescence (F) withrespect to temperature (T). This derivative is plotted as −dF/dT v. Tand converts the melting curves to melting peaks.

EXAMPLE 16

The purified hepatitis B and beta-globin gene fragments of Example 14were melted individually and together with a temperature transition rateof 0.2° C./sec and other conditions as specified in Example 14 (FIG.41). The somewhat subjective determination of Tm from the melting curves(top) is easily called by eye from the melting peaks (bottom). The areaunder the melting peaks can also be quantified by integration of thearea under the curves. The fluorescence baseline was first subtractedfrom the −dF/dT v. T plot assuming that the magnitude of the baselinevaries as the area under the curve. Then the peaks were fit by nonlinearleast squares regression to gaussians with the mean, standard deviation,and height of the peak as the fit parameters. The area under eachgaussian was taken as the peak area. All calculations were performed inthe LabView programming environment (National Instruments, Austin,Tex.). FIG. 41 shows an example of this conversion of melting curves tomelting peaks. The code for these calculations is included as appendixA.

The ability to distinguish specific product from primer dimer and otherreaction artifacts improves the use of double-strand-specific DNA dyesin the quantification of low initial copy numbers. Relatively largeinitial template copy numbers have been quantified using ethidiumbromide (Higuchi & Watson, supra). However, at low initial copy numbers,the background amplification of primer dimers and other amplificationartifacts interferes with the specific amplification signal. With thepresent invention's ability to differentiate specific products fromnon-specific artifacts, double-strand-specific DNA dyes can be used toquantify low initial template copy numbers. This is advantageous becauseof the simplicity of using these dyes. The double-strand-specific DNAdyes can be used in any amplification and custom fluorescently-labeledoligonucleotides are not necessary. Quantification of very low copynumbers with double-strand-specific DNA dyes requires very goodamplification specificity or, as provided by the present invention, ameans to differentiate the desired product from nonspecificamplification.

EXAMPLE 17

The present invention's approach to product purity determination wasused to improve quantitative PCR based on once-per-cycle monitoring ofdouble-strand-specific DNA dye fluorescence. Fluorescence was acquiredonce each cycle after polymerase extension of the product for a seriesof reactions varying in the initial concentration of purifiedbeta-globin template (see FIG. 42A). The beta globin template andamplification conditions were as given in Example 7. The log-linearincrease above background fluorescence began at a cycle number dependenton initial template concentration. The plots of the five reactionsranging from 10⁶ to 10² copies per reaction were separated by about fourcycles. The sample with an average 10² copies per reaction showed adecrease in reaction efficiency, and reactions with initial copy numberbelow 100 gave fluorescence profiles that were less useful. Thefluorescence profiles for the reactions containing 10 and 1 (average)copies rise in reverse order, and the negative control showedamplification after about 30 cycles. This is due to amplification ofprimer dimers and other nonspecific amplification products that cannotbe distinguished from the intended product by once-per-cyclefluorescence monitoring of double-strand-specific DNA specific dyes.

Melting peaks were acquired for each sample (FIG. 42B) and these werefound to correlate well with electrophoresis results (FIG. 42C). Thereaction containing zero and one average initial template copiesproduced no discernible electrophoresis band at the expected 536 basepair location. The reactions containing 10 and 100 initial copies oftemplate showed weak electrophoresis bands. This agreed well with themelting peak analysis, which showed no DNA melting in the range of theintended product (90-92° C.) for the reactions containing zero and oneinitial copies and small peaks in this temperature range for 10 and 100copies. Strong electrophoresis bands for the reactions containing10³-10⁶ initial copies correlate well with large melting peaks in theexpected 90-92° C. range.

The ratio of intended product to total product, determined by meltingpeak integration, ranged from 0.28 for 10⁵ copies to 0.0002 for zeroinitial template copies. Each fluorescence value in FIG. 41A wasmultiplied by the appropriate ratio to give the corrected plot(designated “corrected fluorescence” in FIG. 42D). This procedureextended the effective dynamic range of quantitation to between 10 and 1initial template copies.

Melting peaks can distinguish specific products from non-specificproducts (FIG. 40) and they can distinguish two purified PCR productsmixed together (FIG. 41) so they should also be useful fordistinguishing two specific products amplified together in a singlereaction tube. Melting curves obtained by continuous monitoring of PCRreactions according to the present invention are useful in multiplexPCR.

EXAMPLE 18

In this example, two gene fragments were simultaneously amplified fromgenomic DNA and monitored with SYBR™ Green I fluorescence. During eachamplification cycle, different amplification products denature atmelting temperatures dependent on the length of the product, GC ratio,and other factors well known in the art. The temperature at which eachproduct melts can be monitored with the double-strand-specific dye,SYBR™ Green I. At 81 base pair fragment from the cystic fibrosis genewas amplified using the primers described herein as SEQ ID NO:14 and SEQID NO:15 along with a 98 base pair fragment of the c-erbB-2 (HER2/neu)oncogene using the primers described herein as SEQ ID NO:16 and SEQ IDNO:17.

Amplification reactions were comprised of 50 mM Tris-HCl, pH 8.3, 3 mMMgCl₂, 500 μg/ml of bovine serum albumin, 200 μM of each dNTP, and 0.5μM of the cystic fibrosis primers, 0.3 μM of the HER2/neu primers, a1:30,000 dilution of SYBR™ Green I, 1 U AmpliTaq Gold DNA polymerase(Perkin Elmer, Foster City, Calif.), and 50 ng of human genomic DNA in10 μl.

After activation of the polymerase at 95° C. for 30 minutes, the sampleswere cycled at 94° C. for 0 seconds (slope=20), 55° C. for 0 seconds(slope=20), and 70° C. for 10 seconds (slope=20) for 35 cycles. Thesamples were cooled to 70° C., and the fluorescence was continuouslyacquired during a 0.2° C./sec ramp to 94° C. Melting curves (FIG. 43)clearly showed two distinct products melting at 78° C. (CFTR) and 88° C.(neu). The two products differ in Tm by approximately 10° C. and areeasily distinguishable.

Multiplex amplification is useful in cases where an internal control isneeded during amplification. For example, many translocations aredetectable by PCR by placing primers on each side of the breakpoint. Ifno amplification occurs, the translocation is not present as long as theDNA is intact and no inhibitor is present. These possibilities can beruled out by amplifying a positive control locus in the same reactionmixture. Such control amplifications are best done as internal controlswith simultaneous amplification and detection.

EXAMPLE 19

In this example, the procedure of Example 18 was followed except thatafter activation of the polymerase at 95° C. for 30 minutes, the sampleswere cycled at 94° C. for 0 seconds (slope=20), 55° C. for 0 seconds(slope=20), and 70° C. for 10 sec (slope=20) for 20 cycles, followed by94° C. for 0 seconds (slope=1), 55° C. for 0 seconds (slope=20), and 70°C. for 20 seconds (slope=20) for 15 cycles. For cycles 26-31,fluorescence was continuously acquired during each 1° C./sec transitionfrom 70° C. to 94° C. The melting curves were converted to melting peaksand displayed (FIG. 44).

Note that the amplification efficiency of the CFTR fragment appearsgreater than the neu fragment. The amplification efficiency can berigorously determined by integrating the melting peak data as in Example16.

This kind of quantitative data referenced to a control has manyapplications. For instance, certain oncogenes, such as HER2/neu, areamplified in vivo in many tumors. That is, the genes are replicated ingenomic DNA, sometimes many fold. Often, the clinical behavior of thetumor depends on the degree of oncogene replication. Amplification ofthe oncogene and a control template allows quantitative assessment ofthe relative copy number. As a further example, quantification of viralload in patients infected with HIV or hepatitis C is important inprognosis and therapy. Using a control template and monitoring theefficiency of amplification of both control and natural templates duringamplification, accurate quantification of initial template copy numberis achieved.

The present invention's feature of using melting curves for relativequantification will now be explained. In accordance with the presentinvention, an additional use for melting curves is quantitative PCR.FIG. 42 showed there was a correlation between the area under themelting peak and the amount of specific product. Relative quantificationof two PCR products would be possible if the two products were amplifiedwith similar efficiency (or if the differing efficiencies were known andcompensated for). Relative quantification of two products by integratingmelting peak areas (see Example 16) is an aspect of the currentinvention.

EXAMPLE 20

The cystic fibrosis and HER-2-neu gene fragments of Example 18 wereamplified, purified as in Example 2. and adjusted to 175 μg/ml. Thesamples were mixed in various ratios (total 8 μl) and added to buffer (1μl) and SYBR™ Green I (1 μl). Final concentrations were 50 mM Tris, pH8.3, 3 mM MgCl₂, 250 μg/ml bovine serum albumin, and a 1:30,000 dilutionof SYBR™ Green I. Melting curves were acquired at 0.2° C./sec,background fluorescence subtracted and the peaks integrated as describedin Example 16. The results are displayed in FIG. 45. Excellentcorrelation was found between the relative areas under melting peaks andthe relative amounts of the two products.

Relative quantification of two PCR products is important in manyquantitative PCR applications. Multiplex amplification of two or moreproducts followed by integration of the areas under the melting peakswill be extremely useful in these areas. mRNA is often quantifiedrelative to the amount of mRNA of a housekeeping gene.

Another important use of relative quantification is in competitivequantitative PCR. Typically a competitor is synthesized that has thesame priming sites, but differs in length from the original targetsequence. Known amounts of the competitor are spiked into an unknownsample and relative quantitation is performed. Competitors can be madethat differ from the target sequence in Tm rather than length. Therelative amounts of the products can be quantified by comparing theareas under their melting peaks. As the amount of one of the products isknown, the quantity of the original target can be obtained. Using themelting peak method is significantly easier than the currently usedmethods which involve running multiple tubes for each unknown sample andoften pulling tubes at various cycle numbers during the reaction to findthe log-linear portion of the reaction. The relative amounts of the twoproducts must then be determined. Usually this is done by labeling oneof the dNTPs with a radioisotope and then quantifying the amount oflabel incorporated into each band after agarose gel electrophoresis. Incomparison, the current invention allows the reaction to be monitoredcontinuously so the log-linear portion of the amplification can beeasily identified. Relative quantification can be done quickly byintegration of melting peaks. An all day process is reduced to less thanan hour.

From the foregoing discussion, it will be appreciated that fluorescencemonitoring during DNA amplification is an extraordinarily powerfulanalytical technique. When sequence-specific detection andquantification are desired, resonance energy transfer probes can be usedinstead of double-strand-specific DNA dyes. The Tm of hybridizationprobes shifts about 4-8° C. if a single base mismatch is present. If ahybridization probe is placed at a mutation site, single base mutationsare detectable as a shift in the probe melting temperature.

EXAMPLE 21

The factor V Leiden mutation is a single base change (G to A) thatsubstitutes a glutamine residue for an arginine residue at amino acidresidue 506 (R506Q). For further information, see R. M. Bertina et al.,Mutation in Blood Coagulation Factor V Associated with Resistance toActivated Protein C, 369 Nature 64-67 (1994) and J. Voorberg et al.,Association of Idiopathic Venous Thromboembolism with a SinglePoint-Mutation at Arg⁵⁰⁶ of Factor V, 343 Lancet 1535-36 (1994), both ofwhich are hereby incorporated by reference. As used herein, “factor VLeiden mutation locus” means the nucleotide position in the factor Vgene at which a guanine base in the wild type is replaced by an adeninebase in the factor V Leiden mutant. SEQ ID NO:9 shows a portion of thewild type factor V gene, and SEQ ID NO:10 shows the correspondingportion of the factor V Leiden gene, with the relevant nucleotide atposition 31 in each case. The complete nucleotide sequence of the factorV gene is described at R. J. Jenny et al., Complete cDNA and DerivedAmino Acid Sequence of Human Factor V, 84 Proc. Nat'l Acad. Sci. USA4846-50 (1987), hereby incorporated by reference, and sequences can alsobe obtained at Genbank locus HUMF10. The amino acid change in the mutantfactor V protein makes this clotting factor resistant to degradation andincreases the tendency to clotting and thrombosis. As the most commoncause of inherited thrombophilia, this mutation is the target of acommon laboratory test done in clinical molecular genetics laboratories.

The standard method of analysis for the factor V Leiden mutation is toamplify the gene segment by PCR, digest the resulting amplified productswith a restriction endonuclease that cuts the wild type sequence but notthe mutant, and distinguish digested wild type and undigested mutantproducts by gel electrophoresis. R. M. Bertina et al., supra. This is amethod well known in the art for analysis for defined mutations. Such atest usually requires about 4 hours, including PCR amplification (2hours), enzyme digestion (1 hour), and electrophoresis (1 hour).Post-amplification steps include opening the sample tube, adding theenzyme, and transferring the digested sample to the electrophoresisapparatus. Post-amplification processing increases the risk of endproduct contamination, and manual handling requires care to preventmislabeling of samples. A method that simultaneously amplifies andanalyzes for point mutations would eliminate these concerns.

A method for complete amplification and analysis of the factor V Leidenmutation within 30 min in the same instrument comprises asymmetricallyamplifying a portion of a human genomic DNA sample containing themutation locus, followed by obtaining and analyzing a melting curve forthe amplified DNA. Genomic DNA is prepared according to methods wellknown in the art, e.g. J. Sambrook et al., Molecular Cloning: ALaboratory Manual (2d ed., 1989), hereby incorporated by reference.Preferably, the melting curve is obtained by the resonance energytransfer methodology described above with a fluorogenic hybridizationprobe. Such an assay easily discriminates between homozygous wild type,homozygous mutant, and heterozygous genotypes. In a preferredembodiment, the oligonucleotide probe is 3′-labeled with fluorescein anddesigned to hybridize on the amplified DNA near to a Cy5-labeled primerfor resonance energy transfer. This method can be applied to any definedmutation.

The probe oligonucleotide is preferably about 15-40 nucleotide residuesin length. The probe could conceivably contain as few as about 10nucleotide residues, however, possible disadvantages of such shortoligonucleotides include low specificity, low melting temperature, andincreased background. Oligonucleotides larger than 40 residues couldalso be used, but would be unnecessarily expensive. Thus, the limits onthe size of the probe oligonucleotide are only those imposed byfunctionality. The probe oligonucleotide should span the mutation, butthe mutation preferably does not correspond to either the 5′- or3′-terminal nucleotide residue of the probe. Since the present inventionis based on melting curves, and lack of base pairing at the termini isknown to have less of an effect on melting properties than at internalsites, the probe should be designed such that the mutation occurs at aninternal position.

The oligonucleotide primers for amplification of the selected mutationlocus are preferably about 15 to 30 residues in length. Primers shorterthan the preferred range could be used but may not be as specific aswould be desired. Similarly, primers longer than the preferred rangecould be used, but would be unnecessarily expensive. Thus, the limits onthe sizes of the PCR primers are only those imposed by functionality.

The distance between the resonance energy transfer pair is alsoimportant for the proper functioning of the invention. The optimumdistance between the resonance energy transfer pair is about 5nucleotides. A distance of about 2 to 8 nucleotides is preferred,although a distance of up to about 10-15 nucleotides is functional.Having the resonance energy transfer pair on adjacent nucleotides is notnecessarily beneficial because the distance between the resonance energytransfer pair is effected by the position on the DNA helix.

In this example, PCR amplification was carried out in 10 μl reactionmixtures comprising 50 mM Tris, pH 8.3, 3 mM MgCl₂, 500 μg/ml bovineserum albumin, 200 μM each dNTP, 0.5 μM Cy5-labeled primer (SEQ IDNO:11), 0.2 μM unlabeled opposing primer (SEQ ID NO:12), 0.1 μMfluorescein-labeled probe (SEQ ID NO:13), 0.4 U Taq polymerase, andfifty ng human genomic DNA. Four different samples of DNA were tested:human genomic DNA from an individual homozygous for the factor V Leidenmutation; human genomic DNA from a heterozygous individual; humangenomic DNA from an individual homozygous for the wild type factor Vallele; and a negative control without DNA. The orientation of theCy5-labeled primer, the fluorescein-labeled probe, and the mutation site(identified by asterisk) are shown below:

         Cy5          | 5′-TAATCTGTAAGAGCAGATCC-3′ (SEQ ID NO:l1)                                 *   TAATCTGTAAGAGCAGATCCCTGGACAGGCGAGGAATACAGGTATT (SEQ ID NO:9)  ATTAGACATTCTCGTCTAGGGACCTGTCCGCTCCTTATGTCCATAA                      3′-CTGTCCGCTCCTTATGTCCATAA-5′ (SEQ ID NO:13)                        |                       Fluorescein

The sequence of the unlabeled opposing primer was TGTTATCACACTGGTGCTAA(SEQ ID NO:12) and the amplified product was 186 base pairs in length.The Cy5-labeled primer was obtained as in Example 8. Cycling conditionswere 94° C. for 0 sec (slope=20), 50° C. for 10 sec (slope=20), and 72°C. for 0 sec (slope=1) for 50 cycles, followed by cooling to 45° C. andcontinuous fluorescence monitoring at a slope of 0.2° C./sec to 94° C.for the melting curve. The highest quality melting curves were obtainedat the end of amplification with a slow temperature transition rate(0.2° C./sec—FIG. 46), although monitoring during each cycle at 1°C./sec between 50° C. and 94° C. also provided clear genotypeidentification (FIG. 47). The melting curves are easiest to visualize byplotting the negative derivative of fluorescence with respect totemperature vs temperature (−dF/dT vs T). Such a plot allows facilevisual identification of all possible genotypes from the rawfluorescence data.

The closer the Cy5 label is to the primer's 3′-end, the greater theresonance energy transfer signal. However, the 3′-end must have a free3′-hydroxyl for polymerase extension, and placing the Cy5 too close tothe 3′-end (either on the 3′ or penultimate base) may inhibit polymeraseattachment and extension. The 3′-fluorescein probe should hybridize asclose to the primer as possible (minor overlap of 1-3 bases can betolerated) and the mutation site should be near the middle of the probe.A 5-base separation between the hybridized fluorophores and a mutationat base 8 of a 23-mer probe gave a melting curve shift of 8° C. betweenmutant and wild type sequences (FIG. 46).

Mutation detection by probe melting can also be performed with 2 labeledprobes instead of one labeled probe and one labeled primer. In thisembodiment, one probe is labeled 5′ with Cy5 and the other probe islabeled 3′ with fluorescein. Since both these fluorescent probes can besynthesized directly from the amidites, a manual synthesis step is notrequired as it is in the primer/probe system. The fluorescein-labeledprobe should be designed such that the mutation locus is near the centerof the fluorescein-labeled probe. The length of the Cy5-labeled probeshould be designed such that it melts at a higher temperature (>5° C.)than the fluorescein-labeled probe which spans the mutation locus.Because background from fluorescein is more troublesome than that fromCy5, the concentration of the Cy5-labeled probe should preferably be 2-5fold that of the fluorescein-labeled probe. The two probes shouldhybridize to the same strand of genomic DNA, and the resonance energytransfer pair should be separated by about 0 to 5 nucleotide residues.Alternately, the probe that spans the mutation site can be labeled withCy5 and the other probe labeled with fluorescein.

It will be appreciated that the particular probes and primers disclosedherein for detection of the factor V Leiden mutation are merelyillustrative, and that a person of ordinary skill in the art will beable to design other probes and primers for detection of mutationswithout undue experimentation by following the principles and guidelinesset forth herein. It should also be recognized that although theinvention is described with respect to detection of a single basemutation in genomic DNA, the same principles can be applied to detectionof a mutation in cDNA. Preparation of the cDNA requires extra processsteps and time, as is well known in the art, thus it is preferred to usegenomic DNA because of the advantages of speed and lower cost. Further,the same technique can be used to detect insertions and deletions bydesigning the hybridization probe so that it melting temperature changeswhen the mutation or polymorphism is present. The invention can be usedto detect any known mutation where a probe can be designed to differ inmelting temperature when hybridized to mutant vs wild type.

Although fluorescein and Cy5 were used as resonance energy transferlabels in the example above, other acceptors, such as Cy5.5, can also beused with fluorescein.

EXAMPLE 22

The factor V locus of Example 21 was amplified as before except that theprimer was labeled with Cy5.5 instead of Cy5. Cy5.5 emission wasobserved through a 683 nm long pass dichroic and a 683-703 nm bandpassinterference filter. The Cy5.5 to fluorescein ratio increased abovebackground at about cycle 30 and the ratio approximately doubled by 50cycles of asymmetric amplification. When amplified with wild type DNA,the probe Tm was 65-66° C. as judged by melting peaks.

Another example for detecting single base mutations will now be given.

EXAMPLE 23

There is a common point mutation in the methylenetetrahydrofolatereductase (MTHFR) gene (C₆₇₇T) that converts an alanine to a valineresidue and results in a thermolabile enzyme. This mutation can reduceMTHFR activity and lead to elevated homocysteine plasma levels which hasbeen implicated as an independent risk factor for early vascular diseaseand thrombosis as is well known in the art. One of the primers waslabeled with Cy5 (TGAAGGAGAAGGTGTCT^(*)GCGGGA) (SEQ ID NO:25) where T*represents a modified T residue linked to Cy5 (see Example 9 forsynthesis and purification). The probe sequence wasfluorescein-CCTCGGCTAAATAGTAGTGCGTCGA (SEQ ID NO:26) and the otherprimer was AGGACGGTGCGGTGAGAGTG (SEQ ID NO:27). A 198 base pair fragmentof the MTHFR gene was amplified from 50 ng of human genomic DNA in 50 mMTris, pH 8.3, 2 mM MgCl₂, 500 μg/ml bovine serum albumin, 0.2 mM of eachdNTP, 0.5 μM of the Cy5-labeled primer, 0.1 μM of the opposing primer,0.1 μM of the fluorescein-labeled probe, and 0.4 U Taq DNA polymeraseper 10 μl. Each cycle was 30 sec long and consisted of denaturation at94° C. followed by a 20 sec combined annealing/extension step at 60° C.The temperature transition rate between steps was 20° C./sec. After 60cycles, a melting curve was acquired as follows: heating from 50 -65° C.at 0.5° C./sec, 65-75° C. at 0.1° C./sec, and 75-94° C. at 0.5° C./sec.After baseline subtraction and conversion to melting peaks, all possiblegenotypes were easily distinguished (FIG. 48).

The discriminatory power of hybridization probes is also used to greatadvantage in multiplex or competitive PCR. For example, an artificialtemplate is designed with a single internal base change and ahybridization probe designed to cover the base change as in Examples 21and 23. Relative amplification of the competitor and natural templateare determined by acquiring and integrating melting peaks as in Example16. Alternately, if multiple detection probes are used that sequentiallymelt off different targets at different temperatures, relativequantification is achieved by the same analysis. In general, anyquantitative technique described previously for double-strand-specificDNA dyes can be made sequence specific with hybridization probes.

Absolute Product Concentration by Product Reannealing Kinetics. Productconcentration determinations are also advantageously carried out usingthe present invention. Continuous monitoring of double stranded DNAformation allows DNA quantification at any cycle of amplification byreannealing kinetics. The sample temperature is quickly dropped from thedenaturation temperature and held constant at a lower temperature thatis still high enough to prevent primer annealing (FIG. 2). The rate ofproduct reannealing follows second order kinetics (see B. Young & M.Anderson, Quantitative analysis of solution hybridization, In: NucleicAcid Hybridization: A Practical Approach 47-71 (B. Hames & S. Higgins,eds., (1985), which is now incorporated herein by reference). For anygiven PCR product and temperature, a second order rate constant can bemeasured. Once the rate constant is known, any unknown DNA concentrationcan be determined from experimental reannealing data. Cooling is neverinstantaneous, and some reannealing occurs before a constant temperatureis reached. Rapid cooling will maximize the amount of data available forrate constant and DNA concentration determination. The techniquerequires pure PCR product, but such can be assured by melting curvesalso obtained during temperature cycling using the present invention.This method of quantification by the present invention is advantageouslyindependent of any signal intensity variations between samples.

EXAMPLE 24

A 536 base pair fragment of the beta-globin gene was amplified fromhuman genomic DNA (Example 7) and purified (see Example 2). Differentamounts of the purified DNA were mixed with a 1:30,000 dilution of SYBR™Green I in 5 μl of 50 mM Tris, pH 8.3 and 3 mM MgCl₂. The samples weredenatured at 94° C. and then rapidly cooled to 85° C. The fluorescenceat 520-550 nm was monitored at 85° C. over time. When differentconcentrations of DNA were tested, the shape of the reannealing curvewas characteristic of the DNA concentration (See FIG. 49). For any givenPCR product and temperature, a second order rate constant can bedetermined. FIG. 50 shows the determination of a second orderreannealing rate constant for 100 ng of the 536 base pair fragment in 5μl at 85° C. The curve was fit by non-linear least squares regressionwith F_(max), F_(min), t₀ and k as the floating parameters using thesecond order rate equation shown in FIG. 50. Analysis programs for thiskind of curve fitting are well known in the art (for example, the userdefined curve fit of Delta Graph, DeltaPoint, Inc, Monteray, Calif.).Once the rate constant is known, an unknown DNA concentration can bedetermined from experimental reannealing data.

With the rate constant (k) defined, DNA concentrations are determined onunknown samples. The fluorescence vs time curves of unknown samples arefit by non-linear least squares regression, preferably duringtemperature cycling in real time (for example, using the nonlinearLevenberg-Marquardt method described in the LabView programmingenvironment, National Instruments, Austin, Tex.). For this fit, F_(max),F_(min), t₀, and [DNA] are the floating parameters and k is constant.

Since some fluorescent dyes affect reannealing in a concentrationdependent manner, the assumption of second order kinetics for productreannealing is checked by determining the rate constant at differentstandard DNA concentrations. The relationship is defined and alternateformula for fitting incorporated as necessary.

Also within the scope of the present invention is to use probe annealingrates to determine product concentrations. The rate of fluorescenceresonance energy transfer is followed over time after a quick drop to aprobe annealing temperature that is greater than the primer annealingtemperature (FIG. 2). For the case of amplification with a labeledprimer and one labeled probe, the rate of annealing (and fluorescencegeneration) is second order. When using two labeled probes, the rate offluorescence development is third order. These two arrangements areshown in FIG. 18. When the concentration of the probe(s) is much greaterthan the product concentration, pseudo-first order and pseudo-secondorder equations are adequate to describe the possibilities. Theappropriate rate equations for these different conditions are well knownin the art (see Young, B. and Anderson, M., supra). For the purposes ofthis invention, it is adequate that the prior art suggests appropriaterate equations that are tested experimentally and corrected ifnecessary.

When probe annealing rates are used to determine product concentrations,possible interfering effects include product reannealing (with probedisplacement by branch migration) and primer annealing and extensionthrough the probe. The later is minimized when the probe Tm's are higherthan the primer Tm's and a probe annealing temperature is chosen tominimize primer annealing. FIG. 13 shows that even if extension occurs,the fluorescence increases with time for about 20 sec. During thisperiod, the fluorescence increase depends on product concentration.

Probe annealing rates are used to determine product concentrationsimilar to the method described above for determining productconcentration by product reannealing. The steps are summarized asfollows: (1) choosing the appropriate rate equation for the system, (2)running known DNA standards to determine the rate constant, (3) checkingthe validity of the rate equation by comparing different rate constantsderived from different concentrations, and (4) using the rates constantsto determine the DNA concentration of unknowns from their probeannealing data.

Fluorescence Feedback for Control of Temperature Cycling. In contrast toendpoint and cycle-by-cycle analysis, the present invention can alsomonitor fluorescence throughout each temperature cycle. Continuousfluorescence monitoring can be used to control temperature cyclingparameters. The present invention uses fluorescence feedback for realtime control and optimization of amplification. Continuous fluorescencemonitoring of PCR samples containing a double-strand-specific DNA dye orfluorescently labeled oligonucleotide probes can be used to monitorhybridization and melting during individual amplification cycles. Thisinformation can be used by the temperature control algorithms within thetemperature cycling apparatus to improve and customize thermal cyclingconditions. Conventional PCR is performed by programming all cyclingparameters before amplification. With continuous monitoring,determination of temperature cycling requirements can occur duringamplification, based on continuous observation of annealing, extension,and denaturation. The potential benefits of using hybridizationinformation to control temperature cycling include:

1. Ensuring complete denaturation of the PCR product each cycle while:

a. Minimizing exposure to excessively high denaturation temperaturesthus avoiding heat-induced damage to the amplification products andpolymerase. Limiting the time product is exposed to denaturationtemperatures is especially useful for amplification of long products.

b. Increasing reaction specificity by minimizing the denaturationtemperature. This selects against products with a Tm higher than theintended amplification product.

2. Maximizing the amplification efficiency by ensuring adequate time forprimer annealing each cycle while:

a. Minimizing the amount of time required for amplification by allowingno longer than is needed to reach a certain efficiency of primerannealing.

b. Enhancing reaction specificity by minimizing the time at theannealing temperature.

3. Maximizing the amplification efficiency by ensuring adequate time forproduct extension each cycle while:

a. Minimizing the amount of time required for amplification by allowingno longer than needed to complete product extension.

b. Enhancing reaction specificity by selecting against products longerthan the intended amplification product. These would require longer thanthe allotted time to complete product extension.

4. Initiating thermal cycling changes dependent on the level offluorescence obtained or the current efficiency of amplification. Forexample, over-amplification and nonspecific reaction products can beminimized by terminating thermal cycling when the efficiency drops to acertain level. As another example, temperature cycling can be modifiedto initiate slower temperature ramps for melting curve acquisition whenthe fluorescence becomes detectable. This saves time because the slowerramps need not be used on earlier cycles. Other desirable changes maybecome evident on continued practice of the invention.

Control is based on an estimate of reaction parameters from thefluorescence data. The original fluorescence data is either acquired asa change in fluorescence over time (temperature specific rates ofdenaturation, annealing, and extension), a change in fluorescence overtemperature (product or probe Tm), or a change in extent ofamplification (amplification yield and efficiency). These rates, Tm'sand their first and second derivatives are used to determine optimalreaction parameters that include denaturation temperature and time,primer annealing temperature and time, probe annealing temperature andtime, enzyme extension temperature and time, and number of cycles.

Double-strand-specific DNA dyes are used for the control ofdenaturation, control of extension, and to initiate thermal cyclingchanges at a certain amplification level or efficiency. Resonance energytransfer dyes are used for the control of annealing as will be describedafter the following example.

EXAMPLE 25

A commercial fluorescence monitoring thermal cycler (LC24 LightCycler,Idaho Technology Inc., Idaho Falls, Id.) was modified so that thesoftware is no longer programmed with temperature/time setpoints, but isprogrammed to acquire fluorescence values, then to use these values forthermal cycler control.

As depicted in the Functional Block Diagram (FIG. 51), the Run-TimeProgram communicates through serial and DAQ-board interfaces with theLightCycler. This allows high level access to either temperature orfluorescence data and either can be used by the Board-level Software fortemperature control. However, in the current embodiment of theinstrument, only the temperature data is converted into digital form atthe Controller Hardware level. The fluorescence data is sent in analogform through the Digital acquisition board interface, is analyzed by theRun-time Program, and is sent back to the Board-level software via theserial interface.

Product Melting Control

A melting peak was acquired for the intended PCR product and a baselinefluorescence was acquired for the sample containing the reactioncocktail at the temperature at which the product was completely melted.

Each cycle of the reaction then used this fluorescence value as atarget. The approach to product denaturation was made in two stages toovercome the time-lag due to the requirement of sending the fluorescencevalue to a remote computer for analysis, then returning the instructionthat the value had been reached. With each product melting step, thetemperature was increased until the fluorescence reached an intermediatevalue, then the heating power was reduced so that a temperature ramprate of roughly 3°/sec gave the computer time to analyze thefluorescence and signal the thermal cycler that product denaturation hadoccurred.

The resulting temperature/time plot (FIG. 52) shows a characteristicincrease in the melting temperature after cycle 20 as the concentrationof amplification product increases. Product Tm is a function of productconcentration.

Product Annealing/Extension

During an extended hold at a combined annealing/extension temperature,the fluorescence was monitored and this information was used to ensurethat adequate, but not excessive time had been allowed for productextension. The fluorescence was monitored at 10 second intervals, if thefluorescence increased more than a settable ratio (typically 1.00-1.05),then the annealing/extension step was continued. Otherwise, the nextproduct melting step was initiated. The interval of 10 seconds waschosen to give a minimum of 20 seconds at the combinedannealing/extension temperature.

The resulting fluorescence/time plot (FIG. 52) shows a characteristicincrease in the dwell time at the combined annealing/extensiontemperature as the concentration of amplification product grows. As theprimer concentration and polymerase become limiting, more time is neededto complete product extension with each cycle.

Amplification Plateau

At the end of each annealing/extension step, the fluorescence value wasacquired and stored. When this value had increased to 1.2 times thelowest end-cycle fluorescence value and had subsequently stoppedincreasing below a user settable ratio (typically 1.00-1.02), thethermal cycling was terminated. Alternately, a melting-curve acquisitionstep was initiated by entering a slow 0.1-0.2° C./second temperatureramp through the product Tm and monitoring the fluorescence of thesample continuously.

The resulting fluorescence/time plot (FIG. 52) shows that aftertwenty-five cycles of amplification the ratio of cycle-by-cyclefluorescence growth fell below 1.00 and the reaction was terminated.

In one embodiment of the present invention, detection of theamplification plateau is used to acquire a high-resolution meltingcurves for each sample in a multiple sample run at the optimaltemperature cycle for each sample. As a sample reaches its amplificationplateau, a melting-curve is acquired for that sample, then regulartemperature cycling is resumed until another reaction reaches itsamplification plateau.

Real time monitoring and control of annealing distinct from extension isalso provided by the present invention. If one of the primers is3′-labeled with Cy5, no extension can occur. However, if labeled primer(1-10%) is mixed with unlabeled primer (90-99%), amplificationefficiency will be slightly decreased, but annealing is observable asfluorescence energy transfer from a double-strand-specific dye to Cy5.The primer with the lowest Tm (as determined by nearest neighborthermodynamics as known in the art) is labeled with Cy5 and SYBR™ GreenI is included as a double-strand-specific dye. Alternately, primerannealing can be monitored indirectly with equivalent complementaryoligonucleotides. An oligonucleotide of the same length and Tm as thelowest melting primer is designed with no complementarity to theamplified sequence. This oligonucleotide is 5′-labeled with Cy5 and itscomplement is 3′-labeled with fluorescein or some other resonance energytransfer pair. Hybridization of these oligonucleotides is followed byresonance energy transfer. The concentration of one probe is made thesame as the concentration of the lowest Tm primer and the concentrationof the other probe is made much less than this in order to obtainpseudo-first-order kinetics that approximates the pseudo-first-orderkinetics of primer annealing to product. The efficiency of annealing ismonitored and used to control annealing temperature and times by one ofthese methods.

It is also within the scope of the present invention to entirely replacetemperature and time setpoints with fluorescence feedback control. Forexample, three samples are placed in a fluorescence temperature cyclerwith feedback capacity. The samples are:

1. A non-reacting sample containing amplified product and SYBR™ Green I.

2. A non-reacting sample containing complementary fluorescently labeledprimers with a Tm equal to the lowest Tm primer and concentrations asnoted above.

3. The sample to be amplified and SYBR™ Green I.

With each cycle of amplification, product denaturation is ensured bymonitoring sample 1 as the temperature is increased. A melting curve isdetermined in real-time and when the sample has denatured, thetransition to the annealing step is begun. Primer annealing is monitoredindirectly through the hybridization of two complementary primers insample 2. One of the primers is 3′ labeled with fluorescein and theother is 5′ labeled with Cy5 or similar dye. The temperature isdecreased until sample 2 shows primer hybridization as indicated by anincrease in the ratio of fluorescence at 670 nm/540 nm. This ratioincreases due to resonance energy transfer between the fluorophores whenthey are approximated by hybridization. Product extension is followed bymonitoring the fluorescence of one or more of the actual samples asdemonstrated in Example 25.

Summary. From the foregoing discussion, it will be appreciated thatcontinuous fluorescence monitoring during DNA amplification to monitorhybridization is an extraordinarily powerful analytical technique. Usingthe methods described herein and depending on the number of initialtemplate copies present, product identification and quantification canbe achieved in five to twenty minutes after temperature cycling hasbegun. The present invention achieves several advantages not heretoforeavailable in the art. For example, the present invention providessingle-color fluorescence methods to monitor product purity, relativequantitation by multiplex PCR or competitive PCR, absolute productquantification by reannealing kinetics, and an improved method forinitial template quantification by fluorescence vs cycle number plots.The present invention also provides dual-color, sequence-specificmethods for sequence variation detection, relative quantitation bymultiplex PCR or competitive PCR, product quantification by probeannealing kinetics, and initial template quantification by fluorescencevs cycle number plots.

The following table compares double-strand-specific DNA dyes, hydrolysisprobes, and hybridization probes useful in continuous monitoring of PCR.The fluorescence of double-strand-specific DNA dyes depends on thestrand status of the DNA. The dual-labeled hydrolysis probes arequenched while intact and donor fluorescence increases when the probe ishydrolyzed. Hybridization probes depend on increased resonance energytransfer when hybridization brings 2 fluorophores closer together.

Summary of Fluorescent Probes for Continuous Monitoring of PCRFluorescent Probe dsDNA dye Hydrolysis Hybridization Mechanism Strandstatus Quenching Transfer Probe Synthesis Unnecessary Difficult SimpleSpecificity Product Tm Sequence Sequence Melting Analysis Yes No YesMulticolor Analysis No Yes Yes

In accordance with the present invention, time, temperature andfluorescence are acquired 1-10 times every sec and fine details ofproduct and/or probe hybridization are observed during temperaturecycling. With double-strand-specific DNA dyes, the hybridization ofproduct with respect to temperature is used to identify products bymelting curves. In addition, relative product quantification is achievedby multiplex amplification of two or more different products that differin Tm. Further, competitive PCR is performed by altering the sequenceinternal to the common primers so that two or more products havedifferent Tm's. Absolute product quantification is obtained by rapidlycooling the denatured product and observing reannealing kinetics. Thesensitivity of initial template quantification with fluorescence vscycle number plots is increased by analysis of product melting curves tocontrol for nonspecific amplification and curve fitting algorithms.Finally, immediate fluorescence feedback for control of denaturationconditions, elongation times and product yield are obtained bymonitoring product strand status with double-strand-specific DNA dyes.

The ability to monitor probe hybridization with fluorescence duringtemperature cycling is a powerful tool. The present invention providesdual-color fluorescence methods that depend on probe hybridization (nothydrolysis) for sequence-specific detection and quantification duringPCR. The annealing kinetics and melting of hybridization probes providesinformation not available with probes that rely on exonucleasehydrolysis between fluorophores. Continuous monitoring ofsequence-specific probe hybridization can be followed over temperaturechanges by resonance energy transfer. Probe melting occurs at acharacteristic temperature determined by its sequence andcomplementarity to the product. Two schemes have been detailed by thepresent invention, (1) two adjacent hybridization probes, and (2) onelabeled probe that hybridizes to a single stranded PCR product thatincorporates a labeled primer. The melting temperature ofsequence-specific probes identifies and discriminates products duringPCR. DNA polymorphisms or mutations, including single base mutations,are detected by probe Tm shifts. In addition, relative productquantification is achieved by multiplex amplification of at least twodifferent products with one or more probes that melt from theirrespective products at different temperatures. Further, competitive PCRis performed by altering the sequence internal to the primers so thatone or more probes hybridize to the competitor and the natural templateat different Tm's. Alternately, relative or competitive PCR areperformed by multicolor analysis with probes labeled with differentfluorophores, such as Cy5 and Cy5.5. Absolute product concentration isdetermined by analysis of probe annealing kinetics. Initial templatecopy number is determined from fluorescence vs cycle number plots bycurve fitting algorithms.

When multiplex analysis in one PCR reaction is desired, it is commonpractice to use different fluorescent labels with distinguishableemission spectra to identify the multiple products. The analysis iscomplicated by the limited number of fluorophores available and theoverlapping emission spectra of those fluorophores that are available(see H M Shapiro, supra). Analysis of product or probe hybridizationwith melting curves is another method to distinguish multiple PCRproducts. By following hybridization during temperature cycling, thenumber of probes and/or spectral colors needed to distinguish multipleproducts can be minimized.

The present invention may be embodied in other specific forms withoutdeparting from its spirit or essential characteristics. The describedembodiments are to be considered in all respects only as illustrativeand not restrictive. The scope of the invention is, therefore, indicatedby the appended claims rather than by the foregoing description. Allchanges which come within the meaning and range of equivalency of theclaims are to be embraced within their scope.

Programming code for carrying out melting curve and other analyses isfound in the Programming Code Appendix (Microfiche) to U.S. applicationSer. No. 08/869,276, already incorporated by reference.

The following Sequence Listing is identical to the Sequence Listingcontained in U.S. application Ser. No. 08/869,276, filed Jun. 4, 1997.

                   #             SEQUENCE LISTING(1) GENERAL INFORMATION:    (iii) NUMBER OF SEQUENCES: 27(2) INFORMATION FOR SEQ ID NO: 1:      (i) SEQUENCE CHARACTERISTICS:          (A) LENGTH: 20 base  #pairs           (B) TYPE: nucleic acid          (C) STRANDEDNESS: single-s #tranded          (D) TOPOLOGY: linear    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:  #1:CGTGGTGGAC TTCTCTCAAT             #                  #                   # 20 (2) INFORMATION FOR SEQ ID NO: 2:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 20 base  #pairs          (B) TYPE: nucleic acid           (C) STRANDEDNESS: single-s#tranded           (D) TOPOLOGY: linear    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:  #2:AGAAGATGAG GCATAGCAGC             #                  #                   # 20 (2) INFORMATION FOR SEQ ID NO: 3:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 35 base  #pairs          (B) TYPE: nucleic acid           (C) STRANDEDNESS: single-s#tranded           (D) TOPOLOGY: linear    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:  #3:CAAACAGACA CCATGGTGCA CCTGACTCCT GAGGA        #                  #       35 (2) INFORMATION FOR SEQ ID NO: 4:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 30 base  #pairs          (B) TYPE: nucleic acid           (C) STRANDEDNESS: single-s#tranded           (D) TOPOLOGY: linear    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:  #4:AAGTCTGCCG TTACTGCCCT GTGGGGCAAG          #                  #           30 (2) INFORMATION FOR SEQ ID NO: 5:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 27 base  #pairs          (B) TYPE: nucleic acid           (C) STRANDEDNESS: single-s#tranded           (D) TOPOLOGY: linear    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:  #5:TCTGCCGTTA CTGCCCTGTG GGGCAAG           #                  #             27 (2) INFORMATION FOR SEQ ID NO: 6:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 20 base  #pairs          (B) TYPE: nucleic acid           (C) STRANDEDNESS: single-s#tranded           (D) TOPOLOGY: linear    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:  #6:CAACTTCATC CACGTNCACC             #                  #                   # 20 (2) INFORMATION FOR SEQ ID NO: 7:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 18 base  #pairs          (B) TYPE: nucleic acid           (C) STRANDEDNESS: single-s#tranded           (D) TOPOLOGY: linear    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:  #7:CTGTCCGTGA CGTGGATT              #                   #                  #  18 (2) INFORMATION FOR SEQ ID NO: 8:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 18 base  #pairs          (B) TYPE: nucleic acid           (C) STRANDEDNESS: single-s#tranded           (D) TOPOLOGY: linear    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:  #8:AAGTCCTCCG AGTATAGC              #                   #                  #  18 (2) INFORMATION FOR SEQ ID NO: 9:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 46 base  #pairs          (B) TYPE: nucleic acid           (C) STRANDEDNESS: single-s#tranded           (D) TOPOLOGY: linear    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:  #9:TAATCTGTAA GAGCAGATCC CTGGACAGGC GAGGAATACA GGTATT   #                 46 (2) INFORMATION FOR SEQ ID NO: 10:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 46 base  #pairs          (B) TYPE: nucleic acid           (C) STRANDEDNESS: single-s#tranded           (D) TOPOLOGY: linear    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:  #10:TAATCTGTAA GAGCAGATCC CTGGACAGGC AAGGAATACA GGTATT   #                 46 (2) INFORMATION FOR SEQ ID NO: 11:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 20 base  #pairs          (B) TYPE: nucleic acid           (C) STRANDEDNESS: single-s#tranded           (D) TOPOLOGY: linear    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:  #11:TAATCTGTAA GAGCAGATCC             #                  #                   # 20 (2) INFORMATION FOR SEQ ID NO: 12:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 20 base  #pairs          (B) TYPE: nucleic acid           (C) STRANDEDNESS: single-s#tranded           (D) TOPOLOGY: linear    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:  #12:TGTTATCACA CTGGTGCTAA             #                  #                   # 20 (2) INFORMATION FOR SEQ ID NO: 13:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 23 base  #pairs          (B) TYPE: nucleic acid           (C) STRANDEDNESS: single-s#tranded           (D) TOPOLOGY: linear    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:  #13:AATACCTGTA TTCCTCGCCT GTC            #                  #                23 (2) INFORMATION FOR SEQ ID NO: 14:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 20 base  #pairs          (B) TYPE: nucleic acid           (C) STRANDEDNESS: single-s#tranded           (D) TOPOLOGY: linear    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:  #14:ATGCCTGGCA CCATTAAAGA             #                  #                   # 20 (2) INFORMATION FOR SEQ ID NO: 15:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 20 base  #pairs          (B) TYPE: nucleic acid           (C) STRANDEDNESS: single-s#tranded           (D) TOPOLOGY: linear    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:  #15:GCATGCTTTG ATGACGCTTC             #                  #                   # 20 (2) INFORMATION FOR SEQ ID NO: 16:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 20 base  #pairs          (B) TYPE: nucleic acid           (C) STRANDEDNESS: single-s#tranded           (D) TOPOLOGY: linear    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:  #16:CGGATCTTCT GCTGCCGTCG             #                  #                   # 20 (2) INFORMATION FOR SEQ ID NO: 17:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 20 base  #pairs          (B) TYPE: nucleic acid           (C) STRANDEDNESS: single-s#tranded           (D) TOPOLOGY: linear    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:  #17:CCTCTGACGT CCATCATCTC             #                  #                   # 20 (2) INFORMATION FOR SEQ ID NO: 18:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 31 base  #pairs          (B) TYPE: nucleic acid           (C) STRANDEDNESS: single-s#tranded           (D) TOPOLOGY: linear    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:  #18:GAAGTCTGCC GTTACTGCCC TGTGGGGCAA G         #                  #          31 (2) INFORMATION FOR SEQ ID NO: 19:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 26 base  #pairs          (B) TYPE: nucleic acid           (C) STRANDEDNESS: single-s#tranded           (D) TOPOLOGY: linear    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:  #19:CTGCCGTACT GCCCTGTGGG GCAAGG           #                  #              26 (2) INFORMATION FOR SEQ ID NO: 20:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 26 base  #pairs          (B) TYPE: nucleic acid           (C) STRANDEDNESS: single-s#tranded           (D) TOPOLOGY: linear    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:  #20:ATGCCCTCCC CCATGCCATC CTGCGT           #                  #              26 (2) INFORMATION FOR SEQ ID NO: 21:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 20 base  #pairs          (B) TYPE: nucleic acid           (C) STRANDEDNESS: single-s#tranded           (D) TOPOLOGY: linear    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:  #21:CAACTTCATC CACGTTCACC             #                  #                   # 20 (2) INFORMATION FOR SEQ ID NO: 22:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 27 base  #pairs          (B) TYPE: nucleic acid           (C) STRANDEDNESS: single-s#tranded           (D) TOPOLOGY: linear    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:  #22:GTCTGCCGTT ACTGCCCTGT GGGGCAA           #                  #             27 (2) INFORMATION FOR SEQ ID NO: 23:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 32 base  #pairs          (B) TYPE: nucleic acid           (C) STRANDEDNESS: single-s#tranded           (D) TOPOLOGY: linear    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:  #23:CCTCAAACAG ACACCATGGT GCACCTGACT CC        #                  #          32 (2) INFORMATION FOR SEQ ID NO: 24:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 30 base  #pairs          (B) TYPE: nucleic acid           (C) STRANDEDNESS: single-s#tranded           (D) TOPOLOGY: linear    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:  #24:GAAGTCTGCC GTTACTGCCC TGTGGGGCAA          #                  #           30 (2) INFORMATION FOR SEQ ID NO: 25:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 23 base  #pairs          (B) TYPE: nucleic acid           (C) STRANDEDNESS: single-s#tranded           (D) TOPOLOGY: linear    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:  #25:TGAAGGAGAA GGTGTCTGCG GGA            #                  #                23 (2) INFORMATION FOR SEQ ID NO: 26:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 25 base  #pairs          (B) TYPE: nucleic acid           (C) STRANDEDNESS: single-s#tranded           (D) TOPOLOGY: linear    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:  #26:CCTCGGCTAA ATAGTAGTGC GTCGA           #                  #               25 (2) INFORMATION FOR SEQ ID NO: 27:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 20 base  #pairs          (B) TYPE: nucleic acid           (C) STRANDEDNESS: single-s#tranded           (D) TOPOLOGY: linear    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:  #27:AGGACGGTGC GGTGAGAGTG             #                  #                   # 20

What is claimed is:
 1. A method for detecting a target nucleic acid sequence in a biological sample during amplification comprising the steps of: adding a thermostable polymerase and primers configured for amplification of the target nucleic acid sequence to the biological sample; amplifying the target nucleic acid sequence by polymerase chain reaction in the presence of a fluorescent dye selected from the group consisting of SYBR™ Green I and pico green, the polymerase chain reaction comprising thermally cycling the biological sample between at least a denaturation temperature and an elongation temperature through a plurality of amplification cycles using a rapid temperature cycling profile wherein 30 amplification cycles are completed in 10 to 30 minutes; illuminating the biological sample comprising the amplified target nucleic acid sequence with light at a wavelength absorbed by the fluorescent dye; and detecting a fluorescent emission from the fluorescent dye related to the quantity of the amplified target nucleic acid sequence in the sample.
 2. The method of claim 1 wherein during each amplification cycle the sample is held no more than 60 seconds at the elongation temperature.
 3. The method of claim 1 wherein during each amplification cycle the sample is held less than 20 seconds at the elongation temperature.
 4. The method of claim 1 wherein during each amplification cycle the sample is held less than 1 second at the denaturation temperature.
 5. The method of claim 1 wherein the sample is illuminated and fluorescence is detected during each amplification cycle.
 6. The method of claim 5 wherein a fluorescence value is acquired during an extension or a combined annealing/extension phase at each amplification cycle.
 7. The method of claim 1 wherein the sample is illuminated and fluorescence is detected as the temperature is increased, to generate a melting curve.
 8. A method of real time monitoring of amplification of a target nucleic acid sequence in a biological sample, said method comprising the steps of: amplifying the target sequence by polymerase chain reaction in the presence of a quantity of SYBR™ Green I, said polymerase chain reaction comprising the steps of adding the SYBR™ Green I, a thermostable polymerase, and primers for the target nucleic acid sequence to the biological sample to create an amplification mixture and thermally cycling the amplification mixture between at least a denaturation temperature and an elongation temperature during a plurality of amplification cycles; illuminating the mixture with light at a wavelength absorbed by the SYBR™ Green I in at least a portion of the plurality of amplification cycles; and detecting a fluorescent emission from the SYBR™ Green I following sample illumination, said fluorescent emission being related to the quantity of amplified target nucleic acid in the sample.
 9. The method of claim 8 wherein the fluorescent emission is obtained during an extension phase of each of the plurality of amplification cycles.
 10. The method of claim 8 wherein the fluorescent emission is obtained during a combined annealing/extension phase of each of the plurality of amplification cycles.
 11. A method of real time monitoring of amplification of a target nucleic acid sequence in a biological sample, said method comprising the steps of: amplifying the target sequence by polymerase chain reaction in the presence of SYBR™ Green I, said polymerase chain reaction comprising the steps of adding SYBR™ Green I, a thermostable polymerase, and primers for the target nucleic acid sequence to the biological sample to create an amplification mixture and thermally cycling the amplification mixture between at least a denaturation temperature and an elongation temperature during a plurality of amplification cycles under conditions wherein the SYBR™ Green I retains the ability to produce a fluorescent signal related to the quantity of the nucleic acid sequence; illuminating the sample with light at a wavelength absorbed by the SYBR™ Green I, subsequent to at least a portion of the plurality of amplification cycles; and monitoring fluorescent emission from the SYBR™ Green I in the sample as a function of sample temperature to generate a melting curve for the amplified target sequence.
 12. In a method of monitoring the amplification of a nucleic acid in a biological sample during PCR amplification, comprising the steps of forming an amplification mixture comprising the biological sample, a fluorescent entity capable of producing a fluorescent signal related to the amount of nucleic acid present in the sample, a thermostable polymerase, and primers for the nucleic acid, amplifying the target sequence by thermally cycling the amplification mixture through a plurality of thermal cycles, and illuminating the sample and monitoring the fluorescent signal from the fluorescent entity during amplification, the improvement comprising the step of selecting the fluorescent entity from the group consisting of SYBR™ Green I and pico green.
 13. A PCR reaction product mixture comprising an amplified nucleic acid product and SYBR™ Green I in an amount capable of providing a fluorescence signal indicative of the concentration of the amplified nucleic acid product in said mixture, said product mixture prepared by subjecting a PCR amplification mixture comprising the target nucleic acid to be amplified, oligonucleotide primers, a thermostable polymerase, and the SYBR™ Green I, to sufficient thermal cycles to amplify the target nucleic acid.
 14. A kit for analysis of a nucleic acid sequence during amplification, the kit comprising: an amplification solution comprising a fluorescent dye selected from the group consisting of SYBR™ Green I and pico green; a thermostable DNA polymerase; and deoxynucleoside triphosphates.
 15. The kit of claim 14 further comprising a pair of primers for amplifying the nucleic acid sequence.
 16. A method for detecting a target nucleic acid sequence in a biological sample during amplification comprising the steps of: adding a thermostable polymerase and primers configured for amplification of the target nucleic acid sequence to the biological sample; amplifying the target nucleic acid sequence by polymerase chain reaction in the presence of a fluorescent dye selected from the group consisting of SYBR™ Green I and pico green, the polymerase chain reaction comprising thermally cycling the biological sample between at least a denaturation temperature and an elongation temperature through a plurality of amplification cycles; illuminating the biological sample comprising the amplified target nucleic acid sequence with tight at a wavelength absorbed by the fluorescent dye; and detecting a fluorescent emission from the fluorescent dye related to the quantity of the amplified target nucleic acid sequence in the sample; wherein during each of the plurality of amplification cycles the sample is held no more than 60 seconds at the elongation temperature and held less than 1 second at the denaturation temperature. 